Oxazolidin-2-one compounds and uses thereof

ABSTRACT

The present invention relates to oxazolidin-2-one substituted pyrimidine compounds that act as PI3K (phosphatidylinositol-3-kinase) inhibitors, as well as pharmaceutical compositions thereof, methods for their manufacture and uses for the treatment of conditions, diseases and disorders dependent on PI3K.

FIELD OF THE INVENTION

The present invention relates to oxazolidin-2-one substituted pyrimidinecompounds that act as PI3K (phosphatidylinositol-3-kinase) inhibitors,as well as pharmaceutical compositions thereof, methods for theirmanufacture and uses for the treatment of conditions, diseases anddisorders dependent on PI3K.

BACKGROUND OF THE INVENTION

Phosphatidylinositol 3-kinases (PI3Ks) comprise a family of lipidkinases that catalyze the transfer of phosphate to the D-3′ position ofinositol lipids to produce phosphoinositol-3-phosphate (PIP),phosphoinositol-3,4-diphosphate (PIP₂) andphosphoinositol-3,4,5-triphosphate (PIP₃) that, in turn, act as secondmessengers in signaling cascades by docking proteins containingpleckstrin-homology, FYVE, Phox and other phospholipid-binding domainsinto a variety of signaling complexes often at the plasma membrane((Vanhaesebroeck et al., Annu. Rev. Biochem 70:535 (2001); Katso et al.,Annu. Rev. Cell Dev. Biol. 17:615 (2001)). Of the two Class 1 PI3Ks,Class 1A PI3Ks are heterodimers composed of a catalytic p110 subunit (α,β, δ isoforms) constitutively associated with a regulatory subunit thatcan be p85α, p55α, p50α, p85β or p55γ. The Class 1B sub-class has onefamily member, a heterodimer composed of a catalytic p110γ subunitassociated with one of two regulatory subunits, p101 or p84 (Fruman etal., Annu Rev. Biochem. 67:481 (1998); Suire et al., Curr. Biol. 15:566(2005)). The modular domains of the p85/55/50 subunits include SrcHomology (SH2) domains that bind phosphotyrosine residues in a specificsequence context on activated receptor and cytoplasmic tyrosine kinases,resulting in activation and localization of Class 1A PI3Ks. Class 1BPI3K is activated directly by G protein-coupled receptors that bind adiverse repertoire of peptide and non-peptide ligands (Stephens et al.,Cell 89:105 (1997)); Katso et al., Annu. Rev. Cell Dev. Biol. 17:615-675(2001)). Consequently, the resultant phospholipid products of class IPI3K link upstream receptors with downstream cellular activitiesincluding proliferation, survival, chemotaxis, cellular trafficking,motility, metabolism, inflammatory and allergic responses, transcriptionand translation (Cantley et al., Cell 64:281 (1991); Escobedo andWilliams, Nature 335:85 (1988); Fantl et al., Cell 69:413 (1992)).

In many cases, PIP2 and PIP3 recruit Akt, the product of the humanhomologue of the viral oncogene v-Akt, to the plasma membrane where itacts as a nodal point for many intracellular signaling pathwaysimportant for growth and survival (Fantl et al., Cell 69:413-423 (1992);Bader et al., Nature Rev. Cancer 5:921 (2005); Vivanco and Sawyer,Nature Rev. Cancer 2:489 (2002)). Aberrant regulation of PI3K, whichoften increases survival through Akt activation, is one of the mostprevalent events in human cancer and has been shown to occur at multiplelevels. The tumor suppressor gene PTEN, which dephosphorylatesphosphoinositides at the 3′ position of the inositol ring and in sodoing antagonizes PI3K activity, is functionally deleted in a variety oftumors. In other tumors, the genes for the p110α isoform, PIK3CA, andfor Akt are amplified and increased protein expression of their geneproducts has been demonstrated in several human cancers. Furthermore,mutations and translocation of p85α that serve to up-regulate thep85-p110 complex have been described in human cancers. Finally, somaticmissense mutations in PIK3CA that activate downstream signaling pathwayshave been described at significant frequencies in a wide diversity ofhuman cancers (Kang at el., Proc. Natl. Acad. Sci. USA 102:802 (2005);Samuels et al., Science 304:554 (2004); Samuels et al., Cancer Cell7:561-573 (2005)).

In some tumors the p110δ isoform, PIK3CB is amplified or over-expressed.In addition, studies indicate that tumors driven by PTEN loss may besensitive to p110β rather than p110α. (Jia et al., Nature, 454:776-779(2008). Wee et al., PNAS 105 (35), 13057-13062 (2008); Liu et al.,Nature Rev. Drug Discovery 8:627-644 (2009)).

Both p110δ and p110γ are expressed primarily in the hematopoietic systemand appear to play significant roles in leukocyte signalling (Liu et al.Blood 110(4), 1191-1198 (2007)). However, they do also play roles insome cancers (Knobbe et al., Brain Pathol. 13, 507-518 (2003); Kang etal. PNAS 103(5), 1289-1294 (2006)). p110δ expression is restricted toleukocytes pointing to its potential role in leukocyte-mediated diseases(Vanhaesebroeck et al. PNAS 94(9), 4330-4335 (1997)). p110δ isupregulated in blast cells in patients with acute myeloid leukaemia,where it plays a key role in cell survival (Sujobert et al., Blood106(3), 1063-1066 (2005)) indicating its potential as a target inleukaemia and other haematological malignancies. p110δ activation playsan important role in the development of B-cell malignancies andtherefore inhibition of p110δ could be used to treat B-cell malignanciessuch as chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma(NHL), plasma cell myeloma and Hodgkin's lymphoma (NH) Castillo et al.,Expert Opin. Investig. Drugs 21, 15-22 (2012)).

These observations show that deregulation of phosphoinositol-3 kinaseand the upstream and downstream components of this signaling pathway isone of the most common deregulations associated with human cancers andproliferative diseases (Parsons et al., Nature 436:792 (2005); Hennesseyat el., Nature Rev. Drug Disc. 4:988-1004 (2005)).

Published international patent application WO2007/084786 describessubstituted pyrimidine molecules that inhibit PI3K.

SUMMARY OF THE INVENTION

There remains a need for compounds that inhibit the activity of morethan one of the Class I PI3K isoforms (alpha, beta, delta and gamma),because such compounds are considered to have the ability to avoidadaption mechanisms due to pathway rewiring through the other isoforms,compared to compounds with unique specificity, e.g. specificity for onemember of the PI3K Class I family.

Increased inhibition potency of at least one of the PI3K isoforms (i.e.inhibit at least one PI3K isoform at lower concentrations, especiallyone or both of the alpha and beta isoforms) may also be advantageous. Inthe case of PTEN null tumors, for example, although the driving isoformis p110b, complete efficacy could require participation of the otherClassIA isoforms. There is also a need for compounds which potentlyinhibit PI3Kalpha kinase, e.g. for the treatment of cancers that areprimarily driven by oncogenic forms of the gene encoding p110a (e.g.PIK3CA H1047R or E545K), as well as tumors showing increased copy numberof PIK3CA.

Compounds which show selective inhibition in favour of one or more PI3Kisoforms (for example at least two, preferably three of the alpha, beta,delta and gamma isoforms, e.g. the alpha, beta and delta isoforms)compared to mTOR are also desirable, as the mTOR inhibitory effectgenerally reduces the safety window, more especially when the compoundinhibits mTOR more strongly than PI3K (unfavorable ratio).

Furthermore, PI3K inhibitors which have a reduced, in particular, do notpossess an off-target effect, such as tubulin binding, are desired, assuch effect can cause toxicity effects not connected with the on-targetPI3K inhibition and therefore such compounds may require additionalcareful dosing control to ensure the therapeutic effect is controllableand attributable to PI3K inhibition. Hence there is a need for compoundswhich have a reduced or weak off-target effect or do not have off-targeteffect.

Desirably compounds displaying an improved inhibition of at least one(e.g. PI3Kalpha), but especially two (e.g. PI3Kalpha and PI3Kbeta) orthree (e.g. PI3Kalpha, PI3Kbeta and PI3Kdelta), or all four class 1PI3Ks (PI3Kalpha, PI3Kbeta, PI3Kdelta and PI3Kgamma) as well as areduced (in particular, an absence of) off-target effect are sought.

The present invention provides compounds and pharmaceutical compositionsthereof which compounds are PI3K inhibitors. The invention also providescombinations comprising those compounds. The invention further providesthe compounds of the invention for use in methods of treating,preventing or ameliorating a PI3K mediated disease such as cancer,comprising administering to a subject in need thereof an effectiveamount of a PI3K inhibiting compound of the invention. The inventionalso provides intermediates useful in the preparation of the compoundsof the invention.

The present invention provides in one aspect a compound of Formula (I)

-   -   wherein,    -   R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium; and

-   -   R² is H;    -   R³ is H; and    -   R⁴ is H, and R⁵ is —CH₃ or —CH₂OH; or    -   R⁴ is —CH₂OH, and R⁵ is H;        or    -   R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;    -   R³ is H; and    -   R⁴ is —CH₃, —CH₂OH, —CH₂CH₂OH, —CH₂CH(OH)CH₃ or —CH₂C(OH)(CH₃)₂        and R⁵ is H, or    -   R⁴ is H, and R⁵ is —CH₃, —CH₂OH, —CH₂CH(OH)CH₃ or        —CH₂C(OH)(CH₃)₂, or    -   R⁴ is H or —CH₃ and R⁵ is H or —CH₃;        or    -   R³ is H;    -   R⁴ is H;    -   R² and R⁵ are joined and form —(CH₂)₄—;        or    -   R⁴ is H and R⁵ is H; and    -   R² is —CH₂OH, and R³ is —CH₃; or    -   R² is H or —CH₃, and R³ is —CH₂OH;        or    -   R² is H and R⁴ is H; and    -   R³ and R⁵ are joined and form the group

or the group

or

-   -   R³ is H and R⁵ is H; and    -   R² and R⁴ are joined and form the group

or a pharmaceutically acceptable salt thereof.The wavy line indicates the point of attachment of the morpholine andalso where present, the point of attachment of other shown groups, tothe rest of the molecule.

The compounds of formula (I) are considered suitable, for example, to beused in the treatment of diseases dependent on PI3 kinase, especiallyproliferative diseases such as cancer, e.g. tumor diseases.

The invention may be more fully appreciated by reference to thefollowing description, including the mentioned definitions and theconcluding examples. The described embodiments are to be takenindependently, collectively or in any combination unless otherwisestated. As used herein, the terms “including”, “containing” and“comprising” are used herein in their open, non-limiting sense.

Unless specified otherwise, the term “compounds of the presentinvention” or “a compound of the present invention” and the like refersto compounds of formula (I) and subformulae thereof (e.g. formulae (IA)and (IA′)), and salts of the compounds, as well as isotopically labeledcompounds (including deuterium substitutions).

The compounds of the invention have the stereochemistry depicted informula (I) and subformulae thereof unless stated otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the differential Scanning calorimetry graph of the crystallinematerial of Example 10.

FIG. 2 is the powder X-Ray diffraction graph of the crystalline materialof Example 10.

FIG. 3 is the differential Scanning calorimetry graph of the crystallinematerial of Example 18, batch A.

FIG. 4 is the powder X-Ray diffraction graph of the crystalline materialof Example 18, batch A.

FIG. 5 is the differential Scanning calorimetry graph of the crystallinematerial of Example 18, batch B.

FIG. 6 is the powder X-Ray diffraction graph of the crystalline materialof Example 18, batch B.

FIG. 7 is the differential Scanning calorimetry graph of the crystallinematerial of Example 18, batch C.

FIG. 8 is the powder X-Ray diffraction graph of the crystalline materialof Example 18, batch C.

FIG. 9 is the differential Scanning calorimetry graph of the crystallinematerial of Example 18, batch D.

FIG. 10 is the powder X-Ray diffraction graph of the crystallinematerial of Example 18, batch D.

FIG. 11 is the differential Scanning calorimetry graph of thecrystalline material of Example 18, batch E.

FIG. 12 is the powder X-Ray diffraction graph of the crystallinematerial of Example 18, batch E.

DETAILED DESCRIPTION

Various embodiments of the invention are described herein. It will berecognized that features specified in each embodiment may be combinedwith other specified features to provide further embodiments of thepresent invention. Various (enumerated) embodiments of the invention arealso described herein.

The present invention provides in one aspect a compound according toformula (I):

-   -   wherein,    -   R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is H;R³ is H; andR⁴ is H, and R⁵ is —CH₃ or —CH₂OH; orR⁴ is —CH₂OH, and R⁵ is H;orR² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH, —CH₂CH₂OH, —CH₂CH(OH)CH₃ or —CH₂C(OH)(CH₃)₂ and R⁵is H, orR⁴ is H, and R⁵ is —CH₃, —CH₂OH, —CH₂CH(OH)CH₃ or —CH₂C(OH)(CH₃)₂, orR⁴ is H or —CH₃, and R⁵ is H or —CH₃;orR³ is H;R⁴ is H;R² and R⁵ are joined and form —(CH₂)₄—;orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH;orR² is H;R⁴ is H; andR³ and R⁵ are joined and form the group

orR³ is H;R⁵ is H; andR² and R⁴ are joined and form the group

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of that aspect, there is provided a compoundaccording to formula (I) wherein,

-   -   R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is H;R³ is H; andR⁴ is H, and R⁵ is —CH₃ or —CH₂OH; orR⁴ is —CH₂OH, and R⁵ is H;orR² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH, —CH₂CH₂OH, —CH₂CH(OH)CH₃ or —CH₂C(OH)(CH₃)₂ and R⁵is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃;orR³ is H;R⁴ is H;R² and R⁵ are joined and form —(CH₂)₄—; orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH;orR² is H;R⁴ is H; andR³ and R⁵ are joined and form the group

or the group

orR³ is H;R⁵ is H; andR² and R⁴ are joined and form the group

or a pharmaceutically acceptable salt thereof.

In a more preferred embodiment of that aspect, there is provided acompound according to formula (I) wherein,

-   -   R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is H;R³ is H; andR⁴ is H, and R⁵ is —CH₃ or —CH₂OH; orR⁴ is —CH₂OH, and R⁵ is H;orR² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH, —CH₂CH₂OH, —CH₂CH(OH)CH₃ or —CH₂C(OH)(CH₃)₂ and R⁵is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃;orR³ is H;R⁴ is H;R² and R⁵ are joined and form —(CH₂)₄—;orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH;or a pharmaceutically acceptable salt thereof.

In a further preferred embodiment, there is provided a compound ofFormula (I)

-   -   wherein,    -   R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is H;R³ is H; andR⁴ is H, and R⁵ is —CH₃ or —CH₂OH; orR⁴ is —CH₂OH, and R⁵ is H;orR² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃;orR³ is H;R⁴ is H; andR² and R⁵ are joined and form —(CH₂)₄—;orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH,or a pharmaceutically acceptable salt thereof.

In a further alternative preferred embodiment, there is provided acompound according to Formula (I)

-   -   wherein,    -   R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is H;R³ is H; andR⁴ is H, and R⁵ is —CH₃ or —CH₂OH; orR⁴ is —CH₂OH, and R⁵ is H;orR² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃ or —CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃;orR³ is H;R⁴ is H;R² and R⁵ are joined and form —(CH₂)₄—;orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH,or a pharmaceutically acceptable salt thereof.

With respect to formula (I) in any one of the above-mentionedembodiments, the following detailed description is provided.

R^(1a)

In an embodiment, R^(1a) is H.

In another embodiment R^(1a) is —CH₃.

In a preferred embodiment R^(1a) is H.

Further embodiments of the present invention are described below.

In an embodiment,

R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃;orR³ is H;R⁴ is H; andR² and R⁵ is —(CH₂)₄—;orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH,or a pharmaceutically acceptable salt thereof.

In another embodiment,

R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃;orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH,or a pharmaceutically acceptable salt thereof.

In yet a further embodiment,

R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃,or a pharmaceutically acceptable salt thereof.

In yet a further embodiment,

R¹ is

wherein R^(1a) is H or —CH₃R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃,or a pharmaceutically acceptable salt thereof.

In another embodiment,

R¹ is

wherein R^(1a) is H or —CH₃R² is —CH₃ or —CH₂OH;R³ is H;R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃,or a pharmaceutically acceptable salt thereof.

In another embodiment, preferably,

R¹ is

wherein R^(1a) is H or —CH₃R² is —CH₃ or —CH₂OH;R³ is H;R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH and R⁵ is H orR⁴ is H and R⁵ is CH₃ or —CH₂OH,or a pharmaceutically acceptable salt thereof.

In another embodiment, preferably,

R¹ is

wherein R^(1a) is H or —CH₃R² is —CH₃ or —CH₂OH;R³ is H;R⁴ is —CH₃ or —CH₂CH₂OH, andR⁵ is H,or a pharmaceutically acceptable salt thereof.

In an embodiment,

R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃ or —CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃;orR³ is H;R⁴ is H;R² and R⁵ is —(CH₂)₄—;orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH,or a pharmaceutically acceptable salt thereof.

In another embodiment,

R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃ or —CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃;orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH,or a pharmaceutically acceptable salt thereof.

In yet a further embodiment,

R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃ or —CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃,or a pharmaceutically acceptable salt thereof.

In yet a further embodiment,

R¹ is

wherein R^(1a) is H or —CH₃R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃ or —CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃,or a pharmaceutically acceptable salt thereof.

In another embodiment,

R¹ is

wherein R^(1a) is H or —CH₃R² is —CH₃ or —CH₂OH;R³ is H;R⁴ is —CH₃ or —CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃,or a pharmaceutically acceptable salt thereof.

In another embodiment, preferably,

R¹ is

wherein R^(1a) is H or —CH₃R² is —CH₃ or —CH₂OH;R³ is H;R⁴ is —CH₃ or —CH₂OH and R⁵ is H orR⁴ is H and R⁵ is CH₃ or —CH₂OH,or a pharmaceutically acceptable salt thereof.

In another embodiment, preferably,

R¹ is

wherein R^(1a) is H or —CH₃R² is —CH₃ or —CH₂OH;R³ is H;R⁴ is —CH₃, andR⁵ is H,or a pharmaceutically acceptable salt thereof.

In another preferred embodiment,

R¹ is

wherein R^(1a) is H or —CH₃R² is —CH₂OH;R³ is H;R⁴ is —CH₃, andR⁵ is H,or a pharmaceutically acceptable salt thereof.

In an embodiment, compounds of the following formula (IA′) are provided:

wherein R^(1a), R², R³, R⁴ and R⁵, are as described in any of theabove-mentioned embodiments.

In an embodiment, R^(1a) may be hydrogen, thus providing compounds ofthe following formula (IA):

wherein,R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃,or a pharmaceutically acceptable salt thereof.

In an embodiment of compounds of formula (IA),

R² is —CH₃ or —CH₂OH;

R³ is H;

R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, or

R⁴ is H, and R⁵ is —CH₃ or —CH₂OH, or

R⁴ is H or —CH₃ and R⁵ is H or —CH₃,

or a pharmaceutically acceptable salt thereof.

In another embodiment of compounds of formula (IA),

R² is —CH₃ or —CH₂OH;

R³ is H;

R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH and R⁵ is H or

R⁴ is H and R⁵ is CH₃ or —CH₂OH,

or a pharmaceutically acceptable salt thereof.

In another embodiment of compounds of formula (IA),

R² is —CH₃ or —CH₂OH;

R³ is H;

R⁴ is —CH₃ or —CH₂CH₂OH and

R⁵ is H,

or a pharmaceutically acceptable salt thereof.

In another embodiment of compounds of formula (IA),

R² is —CH₃;

R³ is H;

R⁴ is —CH₂CH₂OH, and

R⁵ is H,

or a pharmaceutically acceptable salt thereof.

Alternatively, in an embodiment where R^(1a) may be hydrogen, compoundsof the following formula (IA) are provided:

wherein,R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃ or —CH₂OH, and R⁵ is H, orR⁴ is H, and R⁵ is —CH₃ or —CH₂OH, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃,or a pharmaceutically acceptable salt thereof.

In an embodiment of compounds of formula (IA),

R² is —CH₃ or —CH₂OH;

R³ is H;

R⁴ is —CH₃ or —CH₂OH, and R⁵ is H, or

R⁴ is H, and R⁵ is —CH₃ or —CH₂OH, or

R⁴ is H or —CH₃ and R⁵ is H or —CH₃,

or a pharmaceutically acceptable salt thereof.

In another embodiment of compounds of formula (IA),

R² is —CH₃ or —CH₂OH;

R³ is H;

R⁴ is —CH₃ or —CH₂OH and R⁵ is H or

R⁴ is H and R⁵ is CH₃ or —CH₂OH,

or a pharmaceutically acceptable salt thereof.

In another embodiment of compounds of formula (IA),

R² is —CH₃ or —CH₂OH;

R³ is H;

R⁴ is —CH₃, and

R⁵ is H,

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of compounds of formula (IA),

R² is —CH₂OH;

R³ is H;

R⁴ is —CH₃, and

R⁵ is H,

or a pharmaceutically acceptable salt thereof.

Further embodiments (enumerated) are provided as follows:

Embodiment 1

A compound of Formula (I)

-   -   wherein,    -   R¹ is

wherein R^(1a) is H or —CH₃or R¹ is

wherein D is deuterium;R² and R³ is H;R⁴ is H, and R⁵ is —CH₃ or —CH₂OH; orR⁴ is —CH₂OH, and R⁵ is H;orR² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H;R⁴ is —CH₃, —CH₂OH, —CH₂CH₂OH, —CH₂CH(OH)CH₃ or —CH₂C(OH)(CH₃)₂ and R⁵is H, orR⁴ is H, and R⁵ is —CH₃, —CH₂OH, —CH₂CH(OH)CH₃ or —CH₂C(OH)(CH₃)₂, orR⁴ is H or —CH₃ and R⁵ is H or —CH₃;orR³ is H;R⁴ is H;R² and R⁵ are joined and form —(CH₂)₄—;orR⁴ is H;R⁵ is H; andR² is —CH₂OH, and R³ is —CH₃; orR² is H or —CH₃, and R³ is —CH₂OH;orR² is H;R⁴ is H; andR³ and R⁵ are joined and form the group

or the group

orR³ is H;R⁵ is H; andR² and R⁴ are joined and form the group

or a pharmaceutically acceptable salt thereof.

Embodiment 2

A compound according to Embodiment 1, wherein,

-   -   R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;    -   R³ is H;    -   R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, or    -   R⁴ is H, and R⁵ is —CH₃ or —CH₂OH, or    -   R⁴ is H or —CH₃ and R⁵ is H or —CH₃;    -   or    -   R³ is H;    -   R⁴ is H;    -   R² and R⁵ is —(CH₂)₄—;    -   or    -   R⁴ is H;    -   R⁵ is H; and    -   R² is —CH₂OH, and R³ is —CH₃; or    -   R² is H or —CH₃, and R³ is —CH₂OH,    -   or a pharmaceutically acceptable salt thereof.

Embodiment 3

A compound according to Embodiment 1 or Embodiment 2, wherein

-   -   R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;    -   R³ is H;    -   R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, or    -   R⁴ is H, and R⁵ is —CH₃ or —CH₂OH, or    -   R⁴ is H or —CH₃ and R⁵ is H or —CH₃;    -   or    -   R⁴ is H;    -   R⁵ is H; and    -   R² is —CH₂OH, and R³ is —CH₃; or    -   R² is H or —CH₃, and R³ is —CH₂OH,        or a pharmaceutically acceptable salt thereof.

Embodiment 4

A compound according to any one of Embodiments 1 to 3, wherein

-   -   R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;    -   R³ is H;    -   R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, or    -   R⁴ is H, and R⁵ is —CH₃ or —CH₂OH, or    -   R⁴ is H or —CH₃ and R⁵ is H or —CH₃,        or a pharmaceutically acceptable salt thereof.

Embodiment 5

A compound according to any one of Embodiments 1 to 4, of formula (IA′)

-   -   wherein R^(1a) is H or —CH₃,        or a pharmaceutically acceptable salt thereof.

Embodiment 6

A compound according to Embodiment 1, of formula (IA):

-   -   wherein,    -   R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;    -   R³ is H;    -   R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, or    -   R⁴ is H, and R⁵ is —CH₃ or —CH₂OH, or    -   R⁴ is H or —CH₃ and R⁵ is H or —CH₃,        or a pharmaceutically acceptable salt thereof.

Embodiment 7

A compound according to Embodiment 6, wherein

-   -   R² is —CH₃ or —CH₂OH;    -   R³ is H;    -   R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, or    -   R⁴ is H, and R⁵ is —CH₃ or —CH₂OH, or    -   R⁴ is H or —CH₃ and R⁵ is H or —CH₃,        or a pharmaceutically acceptable salt thereof.

Embodiment 8

A compound according to Embodiment 7, wherein

-   -   R² is —CH₃ or —CH₂OH;    -   R³ is H;    -   R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH and R⁵ is H or    -   R⁴ is H and R⁵ is CH₃ or —CH₂OH,        or a pharmaceutically acceptable salt thereof.

Embodiment 9

A compound according to Embodiment 8, wherein

-   -   R² is —CH₃ or —CH₂OH;    -   R³ is H;    -   R⁴ is —CH₃ or —CH₂CH₂OH and    -   R⁵ is H,        or a pharmaceutically acceptable salt thereof.

Embodiment 10

A compound, or a pharmaceutically acceptable salt thereof, according toEmbodiment 1 which is selected from

-   (S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-methyl-oxazolidin-2-one,-   (S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5,5-dimethyl-oxazolidin-2-one,-   racemic    3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-4,5′-bipyrimidin-6-yl)-4-(hydroxymethyl)-4-methyloxazolidin-2-one,-   (S)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-4,5′-bipyrimidin-6-yl)-4-(hydroxymethyl)-4-methyloxazolidin-2-one    (absolute stereochemistry not determined),-   (R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-4,5′-bipyrimidin-6-yl)-4-(hydroxymethyl)-4-methyloxazolidin-2-one    (absolute stereochemistry not determined),-   (3aS,7aS)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-hexahydro-benzooxazol-2-one,-   (S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-methoxymethyl-oxazolidin-2-one,-   (4S,5S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one,-   (S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-oxazolidin-2-one,-   (4S,5R)-3-(2′-Amino-2-(D8-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one,-   (S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-(2-hydroxy-ethyl)-oxazolidin-2-one,-   (4S,5R)-3-[Z-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-hydroxymethyl-5-methyl-oxazolidin-2-one,-   Formic acid    (4S,5R)-3-(2′-amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-methyl-2-oxo-oxazolidin-4-ylmethyl    ester,-   (S)-3-[Z-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-methyl-oxazolidin-2-one,-   (S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-oxazolidin-2-one,-   (4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-4-methyl-oxazolidin-2-one,-   (S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-methyl-oxazolidin-2-one,-   (S)-3-(2′-amino-2-D8-morpholino-4′-(trifluoromethyl)-[4,5-bipyrimidin]-6-yl)-4-methyloxazolidin-2-one,-   (4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one,-   (4S,5S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-4-methyl-oxazolidin-2-one,-   (R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-oxazolidin-2-one,-   (3aR,6aR)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-tetrahydrofuro[3,4-d]oxazol-2(3H)-one,-   racemic    (3aR*,6R*,6aR*)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-hydroxyhexahydro-2H-cyclopenta[d]oxazol-2-one,-   (3aR,6R,6aR)-(Z-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-hydroxyhexahydro-2H-cyclopenta[d]oxazol-2-one,-   (3aS,6S,6aS)-(Z-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-hydroxyhexa-hydro-2H-cyclopenta[d]oxazol-2-one,    and-   (4S,5R)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-hydroxyethyl)-4-methyloxazolidin-2-one.

Embodiment 11

A compound, or a pharmaceutically acceptable salt thereof, according toEmbodiment 1 which is selected from(4S,5R)-3-[2′-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-hydroxymethyl-5-methyl-oxazolidin-2-one,

-   (4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one,    and-   (4S,5R)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-hydroxyethyl)-4-methyloxazolidin-2-one.

Embodiment 12

A compound which is selected from(4S,5R)-3-[Z-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-hydroxymethyl-5-methyl-oxazolidin-2-one,

-   (4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one,    and-   (4S,5R)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-hydroxyethyl)-4-methyloxazolidin-2-one.

Embodiment 13

The compound(4S,5R)-3-[2′-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-hydroxymethyl-5-methyl-oxazolidin-2-one.

Embodiment 14

The compound(4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one.

Embodiment 15

The compound(4S,5R)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-hydroxyethyl)-4-methyloxazolidin-2-one.

Embodiment 16

A pharmaceutically acceptable salt of the compound of Embodiment 13,Embodiment 14 or Embodiment 15.

In yet another aspect of the present invention, a crystalline form ofthe compound obtained from Example 10 is provided having a X-raydiffraction spectrum substantially the same as the X-ray powderdiffraction spectrum shown in FIG. 2.

In yet another aspect of the present invention, a crystalline form ofthe compound obtained from Example 18, batch A is provided having aX-ray diffraction spectrum substantially the same as the X-ray powderdiffraction spectrum shown in FIG. 4.

In yet another aspect of the present invention, a crystalline form ofthe compound obtained from Example 18, batch B is provided having aX-ray diffraction spectrum substantially the same as the X-ray powderdiffraction spectrum shown in FIG. 6.

In yet another aspect of the present invention, a crystalline form ofthe compound obtained from Example 18, batch C is provided having aX-ray diffraction spectrum substantially the same as the X-ray powderdiffraction spectrum shown in FIG. 8.

In yet another aspect of the present invention, a crystalline form ofthe compound obtained from Example 18, batch D is provided having aX-ray diffraction spectrum substantially the same as the X-ray powderdiffraction spectrum shown in FIG. 10.

In yet another aspect of the present invention, a crystalline form ofthe compound obtained from Example 18, batch E is provided having aX-ray diffraction spectrum substantially the same as the X-ray powderdiffraction spectrum shown in FIG. 12.

The term “essentially the same” with reference to X-ray diffraction peakpositions means that typical peak position and intensity variability aretaken into account. For example, one skilled in the art will appreciatethat the peak positions (2θ) will show some inter-apparatus variability,typically as much as 0.2°. Further, one skilled in the art willappreciate that relative peak intensities will show inter-apparatusvariability as well as variability due to degree of crystallinity,preferred orientation, prepared sample surface, and other factors knownto those skilled in the art, and should be taken as qualitative measureonly.

Specific embodiments are provided by the specific exemplified compoundsdescribed herein.

The present invention provides compounds and pharmaceutical formulationsthereof that are useful in the treatment of diseases, conditions and/ordisorders in which PI3K contributes to the disease pathogenesisdescribed herein.

Compounds of the present invention may be synthesized by syntheticroutes that include processes analogous to those well-known in thechemical arts, particularly in light of the description containedherein. The starting materials are generally available from commercialsources such as Aldrich Chemicals (Milwaukee, Wis.) or are readilyprepared using methods well known to those skilled in the art (e.g.,prepared by methods generally described in Louis F. Fieser and MaryFieser, Reagents for Organic Synthesis, volumes 1-19, Wiley, New York(1967-1999 ed.), or Beilsteins Handbuch der organischen Chemie, 4, Aufl.ed. Springer-Verlag, Berlin, including supplements (also available viathe Beilstein online database)).

For illustrative purposes, the reaction schemes depicted below providepotential routes for synthesizing the compounds of the present inventionas well as key intermediates. For a more detailed description of theindividual reaction steps, see the Examples section below. Those skilledin the art will appreciate that other synthetic routes may be used tosynthesize the inventive compounds. Although specific starting materialsand reagents are depicted in the schemes and discussed below, otherstarting materials and reagents can be easily substituted to provide avariety of derivatives and/or reaction conditions. In addition, many ofthe compounds prepared by the methods described below can be furthermodified in light of this disclosure using conventional chemistry wellknown to those skilled in the art.

In the preparation of compounds of the present invention, protection ofremote functionality (e.g., primary or secondary amino, hydroxyl orcarboxyl groups) of intermediates may be necessary. The need for suchprotection will vary depending on the nature of the remote functionalityand the conditions of the preparation methods. Suitable amino-protectinggroups (NH-Pg) include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC),benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc).Suitable hydroxyl protecting groups include trialkylsilyl ethers whereone or two of the alkyl groups can be replaced by phenyl. Suitablecarboxyl protecting groups (C(O)O-Pg) include alkyl esters (e.g.,methyl, ethyl or t-butyl), benzyl esters, silyl esters, and the like.The need for such protection is readily determined by one skilled in theart. For a general description of protecting groups and their use, seeT. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons,New York, 1991.

Scheme 1 (below) describes a potential route for producing compounds ofFormula (IA′), where R¹-R⁵ are as defined above. In cases where aprotecting group is present a deprotection step is added to convertprotected IA′ into IA′.

Alternatively, compounds of Formula (IA′) can also be synthesized byinverting the steps shown in scheme I, i.e. Suzuki coupling first,followed by the Buchwald reaction with Ib.

For those oxazolidine-2-ones Ib that are not commercially available,Scheme 2 below provides a process for preparing those said intermediateswhere R²-R⁵ are as defined above. If a primary hydroxyl group is presentin either R² to R⁵, a selective protection step is preceding asexemplified in Scheme 3. The amine group can be protected in a furtherpreceding step as shown in Scheme 4, where also a different hydroxylprotecting group is shown.

The protected product of Scheme 3 and be cyclised with triphosgene asgenerally shown in Scheme 2, and specifically shown in Scheme 5, toprovide an example of a Ib intermediate.

The doubly-protected product of Scheme 4 and be cyclised with sodiumhydride as shown in Scheme 6 to provide an example of a Ib intermediate.

Scheme 7 depicts an alternative route to the doubly-protectedintermediate of Scheme 4, which can then be cyclised as already shown inScheme 6.

Scheme 8 below provides the synthesis of the boronic ester intermediateB.

Reaction of the cyclised product of Scheme 6 with the4,6-dichloro-pyrimidine intermediate (e.g. intermediate A or the productfrom step 10.1, both referred to herein below) as shown in Scheme 1, canprovide further intermediates, and specific ones are shown as follows:

Further reaction of the formed intermediates as shown in Scheme 1 withintermediate B, provides a protected product IA′, as follows:

Thus, an intermediate compound of the invention includes a compound ofthe following Formulae:

Wherein R^(1a) and R⁴ are as previously defined herein, Hal is halogen,such as choro, and PG is a protecting group, for example a silylprotecting group forming for example trialkylsilyl ethers where one ortwo of the alkyl groups can be replaced by phenyl, for example analkyl-diphenyl-silyl ether protecting group, specificallydimethyl-tertbutyl-silyl, or diphenyl-tertbutyl silyl.

Another intermediate compound of the invention includes a compound ofthe following Formulae:

Other similarly protected intermediate compounds as depicted hereinabove can be envisaged with reference to the formulae herein if aprimary hydroxyl group is present in either R² to R⁵. Such protectedcompounds are also included in the disclosure. For example where R⁴ isthe group CH₂CH₂OH, this can be protected to provide compounds whereinR⁴ is CH₂CH₂O-PG, wherein PG is as defined above, for example:

Deprotection of the tertiarybutyldiphenylsilyl ortertiarybutyldimethylsilyl protected hydroxyl group (generaldeprotection of silyl ethers) of protected product IA′, to provide, forexample, the final product, can be achieved using HF.pyridine (e.g. inTHF) or HCl.

The compounds of the present invention, or intermediates used herein,may be isolated and used as the compound per se (for example free baseform) or as its salt if for example the pKA value of the compound issuch to allow salt formation. As used herein, the terms “salt” or“salts” refers to an acid addition or base addition salt of a compoundof the invention. “Salts” include in particular “pharmaceuticalacceptable salts”. The term “pharmaceutically acceptable salts” refersto salts that retain the biological effectiveness and properties of thecompounds of this invention and which typically are not biologically orotherwise undesirable. The compounds of the present invention may becapable of forming acid addition salts by virtue of the presence of anamino group. Compounds per se of the invention are preferred.

Inorganic acids and organic acids for formation of pharmaceuticallyacceptable acid addition salts include, e.g., acetate, aspartate,benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate,bisulfate/sulfate, camphorsulformate, chloride/hydrochloride,chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate,gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate,lactate, lactobionate, laurylsulfate, malate, maleate, malonate,mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate,nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate,propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate andtrifluoroacetate salts.

Inorganic acids for salt derivation include, for example, hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like.

Organic acids for salt derivation include, for example, acetic acid,propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,toluenesulfonic acid, sulfosalicylic acid, and the like.Pharmaceutically acceptable base addition salts may be formed withinorganic and organic bases.

Inorganic bases for salt derivation include, for example, ammonium saltsand metals from columns I to XII of the periodic table. In certainembodiments, the salts may be derived from sodium, potassium, ammonium,calcium, magnesium, iron, silver, zinc, and copper; particularlysuitable salts include ammonium, potassium, sodium, calcium andmagnesium salts.

Organic bases for salt derivation include, for example, primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines, basic ion exchange resins,and the like. Certain organic amines include isopropylamine, benzathine,cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazineand tromethamine.

In cases where pharmaceutically acceptable salts of the presentinvention can be formed, they can be synthesized from a parent compound,a basic or acidic moiety, by conventional chemical methods. Generally,such salts can be prepared by reacting free acid forms of thesecompounds with a stoichiometric amount of the appropriate base (such asNa, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like), or byreacting free base forms of these compounds with a stoichiometric amountof the appropriate acid. Such reactions are typically carried out inwater or in an organic solvent, or in a mixture of the two. Generally,use of non-aqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile is desirable, where practicable. Lists ofadditional suitable salts can be found, e.g., in “Remington'sPharmaceutical Sciences”, 20th ed., Mack Publishing Company, Easton,Pa., (1985); and in “Handbook of Pharmaceutical Salts: Properties,Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany,2002).

Unless indicated otherwise, any formula given herein is intended torepresent unlabeled forms. Isotopically labeled forms of the compoundswith deuterium are shown with deuterium (D) as a substituent in place ofH. Other isotopically labeled compounds of the present invention may beprepared and have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine,such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F ³¹P, ³²P, ³⁵S, ³⁶Cl, ¹²⁵I,respectively. The invention can include various isotopically labeledcompounds as defined herein, for example those into which radioactiveisotopes, such as ³H, ¹³C, and ¹⁴C, are present. Such isotopicallylabelled compounds are useful in metabolic studies (with ¹⁴C), reactionkinetic studies (with, for example ²H or ³H), detection or imagingtechniques, such as positron emission tomography (PET) or single-photonemission computed tomography (SPECT) including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Inparticular, an ¹⁸F or labeled compound may be particularly desirable forPET or SPECT studies. Isotopically labeled compounds of this inventioncan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent, for example deuterium labeledmorpholine (D8-morpholine).

Furthermore, substitution with heavier isotopes, particularly deuterium(i.e., ²H or D) may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example increased in vivo half-life,reduced dosage requirements, reduced CYP inhibition (competitive or timedependent) or an improvement in therapeutic index. For example,substitution with deuterium may modulate undesirable side effects of theundeuterated compound, such as competitive CYP inhibition, timedependent CYP inactivation, etc. It is understood that deuterium in thiscontext is regarded as a substituent in compounds of the presentinvention. The concentration of such a heavier isotope, specificallydeuterium, may be defined by the isotopic enrichment factor. The term“isotopic enrichment factor” as used herein means the ratio between theisotopic abundance and the natural abundance of a specified isotope. Ifa substituent in a compound of this invention is denoted deuterium, suchcompound has an isotopic enrichment factor for each designated deuteriumatom of at least 3500 (52.5% deuterium incorporation at each designateddeuterium atom), at least 4000 (60% deuterium incorporation), at least4500 (67.5% deuterium incorporation), at least 5000 (75% deuteriumincorporation), at least 5500 (82.5% deuterium incorporation), at least6000 (90% deuterium incorporation), at least 6333.3 (95% deuteriumincorporation), at least 6466.7 (97% deuterium incorporation), at least6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuteriumincorporation).

Furthermore, the compounds of the present invention, including theirsalts, may also be obtained in the form of their hydrates, or includeother solvents used for their crystallization. The compounds of thepresent invention may inherently or by design form solvates withpharmaceutically acceptable solvents (including water). Such solventmolecules are those commonly used in the pharmaceutical art, which areknown to be innocuous to the recipient, e.g., water, ethanol, and thelike. The term “hydrate” refers to the complex where the solventmolecule is water. The term “solvate” refers to a molecular complex of acompound of the present invention (including pharmaceutically acceptablesalts thereof) with one or more solvent molecules incorporated into thecrystalline lattice structure. The solvent molecules in the solvate maybe present in a regular arrangement and/or a non-ordered arrangement.The solvate may comprise either a stoichiometric or nonstoichiometricamount of the solvent molecules. For example, a solvate with anonstoichiometric amount of solvent molecules may result from partialloss of solvent from the solvate. Solvates may occur as dimers oroligomers comprising more than one molecule or compound according to thepresent invention, within the crystalline lattice structure.

The compounds of the present invention, including salts, hydrates andsolvates thereof, may inherently or by design form polymorphs.

As used herein “polymorph” refers to crystalline forms having the samechemical composition but different spatial arrangements of themolecules, atoms, and/or ions forming the crystal.

As used herein “amorphous” refers to a solid form of a molecule, atom,and/or ions that is not crystalline. An amorphous solid does not displaya definitive X-ray diffraction pattern.

Pharmaceutically acceptable solvates in accordance with the inventioninclude those wherein the solvent of crystallization may be isotopicallysubstituted, e.g. D₂O, d₆-acetone, d₆-DMSO.

It will be recognized by those skilled in the art that the compounds ofthe present invention contain chiral centers and as such exist inisomeric forms. As used herein, the term “isomers” refers to differentcompounds that have the same molecular formula but differ in arrangementand configuration of the atoms. Also as used herein, the term “anoptical isomer” or “a stereoisomer” refers to any of the various stereoisomeric configurations which may exist for a given compound of thepresent invention. It is understood that a substituent may be attachedat a chiral center of a carbon atom. Therefore, compounds of theinvention include enantiomers, shown by indicating stereospecificarrangements at chiral centers in the structural depiction of thecompounds of the invention, wherein a broken wedge bond indicates theattached substituent or atom is below the plane and a solid wedge bondindicates the attached substituent or atom is above the plane.

“Enantiomers” are a pair of stereoisomers that are non-superimposablemirror images of each other. A 1:1 mixture of a pair of enantiomers is a“racemic” mixture. The term is used to designate a racemic mixture whereappropriate.

“Diastereoisomers” are stereoisomers that have at least two asymmetricatoms, but which are not mirror-images of each other.

The absolute stereochemistry is specified according to theCahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer thestereochemistry at each chiral carbon may be specified by either R or S.Resolved compounds whose absolute configuration is unknown can bedesignated (+) or (−) depending on the direction (dextro- orlevorotatory) which they rotate plane polarized light at the wavelengthof the sodium D line. Certain of the compounds described herein containone or more asymmetric centers or axes and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-.

Any asymmetric atom (e.g., chiral carbon or the like) of the compound(s)of the present invention can be enantiomerically enriched, for examplethe (R)- or (S)-configuration. In certain embodiments, each asymmetricatom has at least 50% enantiomeric excess, at least 60% enantiomericexcess, at least 70% enantiomeric excess, at least 80% enantiomericexcess, at least 90% enantiomeric excess, at least 95% enantiomericexcess, or at least 99% enantiomeric excess in the (R)- or(S)-configuration described for the specific asymmetric atom (e.g.chiral carbon).

Accordingly, a compound of the present invention can be in the form of asubstantially pure enantiomer.

Any resulting mixtures of isomers can be separated on the basis of thephysicochemical differences of the constituents, into the pure orsubstantially pure optical isomers, for example, by chromatographyand/or fractional crystallization.

Optically active (R)- and (S)-isomers may be prepared using chiralsynthons or chiral reagents, or resolved using conventional techniques.Any resulting racemates of final products or intermediates can beresolved into the optical antipodes by known methods. For example, knownmethods include separation of the diastereomeric salts thereof, obtainedwith an optically active acid or base, and liberating the opticallyactive acidic or basic compound. In particular, a basic moiety may thusbe employed to resolve the compounds of the present invention into theiroptical antipodes, e.g., by fractional crystallization of a salt formedwith an optically active acid, e.g., tartaric acid, dibenzoyl tartaricacid, diacetyl tartaric acid, di-O,O′-p-toluoyl tartaric acid, mandelicacid, malic acid or camphor-10-sulfonic acid. Racemic products can alsobe resolved by chiral chromatography, e.g., high pressure liquidchromatography (HPLC) using a chiral adsorbent.

If the compound contains a double bond, the substituent may be E or Zconfiguration. If the compound contains a disubstituted cycloalkyl, thecycloalkyl substituent may have a cis- or trans-configuration. Alltautomeric forms are also intended to be included.

Compounds of the invention that contain groups capable of acting asdonors and/or acceptors for hydrogen bonds may be capable of formingco-crystals with suitable co-crystal formers. These co-crystals may beprepared from compounds of the present invention by known co-crystalforming procedures. Such procedures include grinding, heating,co-subliming, co-melting, or contacting in solution compounds of thepresent invention with the co-crystal former under crystallizationconditions and isolating co-crystals thereby formed. Suitable co-crystalformers include those described in WO 2004/078163. Hence the inventionfurther provides co-crystals comprising a compound of the presentinvention.

The compounds of formula (I) inhibit PI3 kinases (PI3K) and maytherefore be useful in the treatment of protein or lipid kinasedependant diseases, especially diseases depending on the class IPI3kinases, PI3Kalpha, PI3Kbeta, PI3Kdelta and PI3Kgamma or one or moreof the individual kinase members thereof or any combination of any twoor more of the mentioned kinases.

Compounds that inhibit the activity of more than one of the Class I PI3Kisoforms (alpha, beta, delta and gamma), in particular substantiallyequipotent on the ClassIA members p110a, p110b and p110d and optionallyas well as the classIB member p110g, are considered to be of benefitbecause such compounds are considered to have the ability to avoidadaption mechanisms due to pathway rewiring through the other isoforms,compared to compounds with unique specificity, e.g. specificity for onemember of the PI3K Class I family. By “equipotent”, it is meant that thecompounds inhibit several isoforms to a comparable extent, e.g. asmeasured in an enzyme or cellular assay described herein.

Increased inhibition potency of at least one of the PI3K isoforms (i.e.inhibit at least one PI3K isoform at lower concentrations) may also beadvantageous. In the case of PTEN null tumors, for example, although thedriving isoform is p110b, complete efficacy could require participationof the other Class IA isoforms. For example potency on the alpha andbeta isoforms could be advantageous.

There is also a need for compounds which potently inhibit PI3Kalphakinase, for example, for the treatment of cancers that are primarilydriven by oncogenic forms of the gene encoding p110a (e.g. PIK3CA H1047Ror E545K), as well as tumors showing increased copy number of PIK3CA.

It is desirable that the compounds of the present invention display thementioned PI3 kinase activity without displaying activity on mTOR, or atleast display a favourable selectivity for inhibiting one or more of theClass I PI3 Kinases over mTOR. For example, compounds which showselective inhibition in favour of one or more PI3K isoforms (for exampleat least two, preferably three, e.g. the alpha, beta and delta isoforms)compared to mTOR are desirable, because the mTOR inhibitory effectgenerally reduces the safety window, more especially when the compoundinhibits mTOR more strongly than PI3K (unfavorable ratio).

Furthermore, PI3K inhibitors which have a reduced off-target effect, ordo not possess an off-target effect, such as not possessing tubulinbinding, are desired, as such effect can cause toxicity effects notconnected with the on-target PI3K inhibition and therefore suchcompounds may require additional careful dosing control to ensure thetherapeutic effect is controllable and attributable to PI3K inhibition.The compounds of the present invention, when measured using theprocedures described herein, show weak or no observable off-targeteffect (tubulin binding).

Compounds that inhibit the activity of more than one of the Class I PI3Kisoforms (alpha, beta, delta and gamma), in particular substantiallyequipotent on the ClassIA members p110a, p110b and p110d and optionallyas well as the classIB member p110g, and in addition have a reducedoff-target effect, or do not possess an off-target effect, such as notpossessing tubulin binding, or reduced tubulin binding, are desired.

Desirably compounds displaying an improved inhibition of at least one(e.g. PI3Kalpha), but especially two (e.g. PI3Kalpha and PI3Kbeta) orthree (e.g. PI3Kalpha, PI3Kbeta and PI3Kdelta), or all four class 1PI3Ks (PI3Kalpha, PI3Kbeta, PI3Kdelta and PI3Kgamma) as well as areduced (in particular, an absence of) off-target effect (e.g. reducedor absence of tubulin binding) are sought. Desirably, these compoundsalso show selective inhibition in favour of one or more PI3K isoforms(for example at least two, preferably three, e.g. the alpha, beta anddelta isoforms) compared to mTOR are desirable.

Consequently, in a further aspect a compound of the present inventionmay be used (e.g. in the manufacture of a medicament) for the treatmentof diseases, conditions or disorders associated with the inhibition orantagonism of the PI3 kinases in a subject (e.g. mammal, preferably ahuman). Because of the relevance to PI3 kinase inhibition, compounds ofthe present invention are therefore considered useful in the treatmentof proliferative diseases such as cancer. Particular diseases/conditionsfor treatment by the compounds of the present invention include a benignor especially malignant tumor, solid tumors, a carcinoma of the brain,kidney, liver, adrenal gland, bladder, breast, stomach (especiallygastric tumors), oesophagus, ovaries, colon, rectum, prostate, pancreas,lung (e.g. non-small cell lung cancer, small cell lung cancer), vagina,thyroid, sarcoma, glioblastomas, multiple myeloma or gastrointestinalcancer, especially colon carcinoma or colorectal adenoma, or a tumor ofthe neck and head, other diseases such as Cowden syndrome,Lhermitte-Duclos disease and Bannayan-Zonana syndrome, (or diseases inwhich the PI3K/PKB pathway is aberrantly activated), prostatehyperplasia, a neoplasia, especially of epithelial character, preferablymammary carcinoma or squamous cell carcinoma, B-cell malignancies suchas chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL),plasma cell myeloma and Hodgkin's lymphoma (NH) or a leukemia. Thecompounds desirably are able to bring about the regression of tumors andto prevent the formation of tumor metastases and the growth of (alsomicro) metastases. It may also be possible to use the compounds offormula (I) in the treatment of diseases of the immune system insofar asseveral or, especially, individual lipid kinases and/or (further)serine/threonine protein kinases are involved.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

The compounds of the present invention are typically used as apharmaceutical composition (e.g., a compound of the present inventionand at least one pharmaceutically acceptable carrier).

Thus, in another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound of the present invention, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

A compound of the present invention may be provided in a composition inamorphous form. A compound of the present invention may be provided in acomposition in its free form, i.e. not in the form of a salt (the freebase form). A compound of the present invention may be provided in acomposition in its free form, i.e. not in the form of a salt (the freebase form) and which is also in amorphous form.

As used herein, the term “pharmaceutically acceptable carrier” includesgenerally recognized as safe (GRAS) solvents, dispersion media,coatings, surfactants, antioxidants, preservatives (e.g., antibacterialagents, antifungal agents), isotonic agents, absorption delaying agents,salts, preservatives, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, buffering agents (e.g., maleic acid, tartaric acid, lactic acid,citric acid, acetic acid, sodium bicarbonate, sodium phosphate, and thelike), and the like and combinations thereof, as would be known to thoseskilled in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Exceptinsofar as any conventional carrier is incompatible with the activeingredient, its use in the therapeutic or pharmaceutical compositions iscontemplated. For purposes of this invention, solvates and hydrates areconsidered pharmaceutical compositions comprising a compound of thepresent invention and a solvent (i.e., solvate) or water (i.e.,hydrate).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent)) is dissolved in a suitable solvent in the presence of one ormore of the excipients described above. The compound of the presentinvention is typically formulated into pharmaceutical dosage forms toprovide an easily controllable dosage of the drug and to give thepatient an elegant and easily handleable product.

The pharmaceutical composition can be formulated for particular routesof administration such as oral administration, parenteraladministration, and rectal administration, etc. In addition, thepharmaceutical compositions of the present invention can be made up in asolid form (including without limitation capsules, tablets, pills,granules, powders or suppositories), or in a liquid form (includingwithout limitation solutions, suspensions or emulsions). Thepharmaceutical compositions can be subjected to conventionalpharmaceutical operations such as sterilization and/or can containconventional inert diluents, lubricating agents, or buffering agents, aswell as adjuvants, such as preservatives, stabilizers, wetting agents,emulsifiers and buffers, etc.

Typically, the pharmaceutical compositions are tablets or gelatincapsules comprising the active ingredient together with

a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine;

b) lubricants, e.g., silica, talcum, stearic acid, its magnesium orcalcium salt and/or polyethyleneglycol; for tablets also

c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone; if desired

d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt,or effervescent mixtures; and/or

e) absorbents, colorants, flavors and sweeteners.

Tablets may be either film coated or enteric coated according to methodsknown in the art.

Solutions of the active ingredient, and also suspensions, and especiallyisotonic aqueous solutions or suspensions, may be used, it beingpossible, e.g., in the case of lyophilized compositions that comprisethe active ingredient alone or together with a carrier, e.g., mannitol,for such solutions or suspensions to be produced prior to use. Thepharmaceutical compositions may be sterilized and/or may compriseadjuvants, e.g., preservatives, stabilizers, wetting and/or emulsifyingagents, solubilizers, salts for regulating the osmotic pressure and/orbuffers; and are prepared in a manner known per se, e.g., by means ofconventional dissolving or lyophilizing processes. The said solutions orsuspensions may comprise viscosity-increasing substances, such as sodiumcarboxymethylcellulose, carboxymethylcellulose,Hydroxypropylmethylcellulose, dextran, polyvinylpyrrolidone or gelatin.

Suspensions in oil comprise as the oil component the vegetable,synthetic or semi-synthetic oils customary for injection purposes. Theremay be mentioned as such especially liquid fatty acid esters thatcontain as the acid component a long-chained fatty acid having from 8-22carbon atoms, especially from 12-22 carbon atoms, e.g., lauric acid,tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, arachidic acid, behenic acid orcorresponding unsaturated acids, e.g., oleic acid, elaidic acid, erucicacid, brasidic acid or linoleic acid, if desired with the addition ofantioxidants, e.g., vitamin E, beta-carotene or3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of those fattyacid esters has a maximum of 6 carbon atoms and is a mono- orpoly-hydroxy, e.g., a mono-, di- or tri-hydroxy; alcohol, e.g.,methanol, ethanol, propanol, butanol or pentanol; or the isomersthereof, but especially glycol and glycerol. The following examples offatty acid esters are therefore to be mentioned: ethyl oleate, isopropylmyristate, isopropyl palmitate, “Labrafil M 2375” (polyoxyethyleneglycerol trioleate, Gattefossé, Paris), “Miglyol 812” (triglyceride ofsaturated fatty acids with a chain length of C₈-C₁₂, Hüls AG, Germany),but especially vegetable oils, such as cottonseed oil, almond oil, oliveoil, castor oil, sesame oil, soybean oil and more especially groundnutoil.

Injectable compositions are prepared in customary manner under sterileconditions; the same applies also to introducing the compositions intoampoules or vials and sealing the containers.

Pharmaceutical compositions for oral administration can be obtained bycombining the active ingredient with solid carriers, if desiredgranulating a resulting mixture, and processing the mixture, if desiredor necessary, after the addition of appropriate excipients, intotablets, dragée cores or capsules. It is also possible for them to beincorporated into plastics carriers that allow the active ingredients todiffuse or be released in measured amounts.

Suitable compositions for oral administration include an effectiveamount of a compound of the invention in the form of tablets, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsion,hard or soft capsules, or syrups or elixirs. Compositions intended fororal use are prepared according to any method known in the art for themanufacture of pharmaceutical compositions and such compositions cancontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets may contain the active ingredient in admixturewith nontoxic pharmaceutically acceptable excipients which are suitablefor the manufacture of tablets. These excipients are, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for example,starch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets are uncoated or coated byknown techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed. Formulations fororal use can be presented as hard gelatin capsules wherein the activeingredient is mixed with an inert solid diluent, for example, calciumcarbonate, calcium phosphate or kaolin, or as soft gelatin capsuleswherein the active ingredient is mixed with water or an oil medium, forexample, peanut oil, liquid paraffin or olive oil.

Pharmaceutical compositions for topical administration can be obtainedby combining the active ingredient with a liquid carrier (e.g. anaqueous liquid carrier) to dissolve or disperse the active, togetherwith further optional formulating ingredients such assolvents/solubilisers, gelling agents, oils, stabilisers, buffers andpreservatives to provide for example a solution, lotion, cream, gel orointment. The pharmaceutical compositions for topical administration maybe provided, for example, for dermal application. The pharmaceuticalcompositions for topical administration may comprise from approximately0.1% to approximately 2% of active ingredient, the active ingredientbeing especially a compound of formula (I), in particular, a compounddescribed in the individual examples herein.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well-known to those skilledin the art and include materials such as bottles (plastic and glass),ampoules, plastic bags, metal cylinders, and the like. The container mayalso include a tamper-proof assemblage to prevent indiscreet access tothe contents of the package. In addition, the container has depositedthereon a label that describes the contents of the container. The labelmay also include appropriate warnings.

The pharmaceutical composition comprising a therapeutically effectiveamount of a compound of the present invention may be formulated for useas a parenteral administration. The pharmaceutical compositions (e.g.,intravenous (iv) formulation) can be subjected to conventionalpharmaceutical operations such as sterilization and/or can containconventional inert diluents, or buffering agents, as well as adjuvants,such as preservatives, stabilizers, wetting agents, emulsifiers andbuffers well known to those of skill in the art.

The present invention further provides anhydrous pharmaceuticalcompositions and dosage forms comprising the compounds of the presentinvention as active ingredients, since water may facilitate thedegradation of certain compounds. Anhydrous pharmaceutical compositionsand dosage forms of the invention can be prepared using anhydrous or lowmoisture containing ingredients and low moisture or low humidityconditions. An anhydrous pharmaceutical composition may be prepared andstored such that its anhydrous nature is maintained. Accordingly,anhydrous compositions are packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (a g., vials),blister packs, and strip packs.

The invention further provides pharmaceutical compositions and dosageforms that comprise one or more agents that reduce the rate by which thecompound of the present invention as an active ingredient willdecompose. Such agents, which are referred to herein as “stabilizers,”include, but are not limited to, antioxidants such as ascorbic acid, pHbuffers, or salt buffers, etc.

A compound of the present invention, in particular, a compound describedin the individual examples herein, may be provided in an amorphous form.

A compound of the present invention, in particular, a compound describedin the individual examples herein may be formulated as a standardsuspension, nanosuspension and solid dispersion, e.g. as follows.

Standard Suspension:

-   -   1.) Required amount of crystalline material of Example 18, Batch        E was weighed with the aim of targetting a formulation        concentration of 3 mg/ml.    -   2.) The crystalline material of Example 18, Batch E was then        dispersed in 0.5% [w/w] Carboxymethylcellulose/0.5% [w/w]        Tween80/Water    -   3.) Suspension was vortexed to homogenize    -   4.) Suspension was sonicated using a probe sonicator to reduce        particle size (2 min)

Nanosuspension:

-   -   1.) 32 mg of crystalline material of Example 18, Batch E        precisely weighed into a tailor-made marble milling device    -   2.) 2.148 g of 0.2 mm Zirconia milling medium was added to the        milling device    -   3.) 0.608 ml of 1% [w/V] HPMC 603 (Hydroxypropylmethylcellulose        grade 603)/0.05% [/w] SDS (SodiumDodecylSulfate)/Water was added        to the milling device    -   4.) The milling devices were closed and put into a rotary mill    -   5.) The sample was milled for 4 h at 400 rpm    -   6.) Nanosuspension were collected using a syringe

Solid Dispersion:

-   -   1.) 30 mg of crystalline material of Example 18, Batch E was        weighed into a lyophilization vial    -   2.) 30 mg of HPMC603 (Hydroxypropylmethylcellulose grade 603)        was added to the same vial    -   3.) 5.6 ml of Dioxane was added to the vial. The vial was closed        with a lid.    -   4.) Sample was stirred at ambient conditions for 12 h    -   5.) Obtained solution was freeze-dried according to the        following conditions

Temperature Time Pressure Temperature [° C.] [min] [ybar] Condenser [°C.] Thermal Treatment −20 30 2500 −40 −20 60 2500 −40 Primary Drying 25180 2000 −40 25 60 500 −40 25 120 100 −40 Post Heat 25 1000 2000 −40

When providing a compound of the invention as a solid dispersion,prepared for example by combining the compound with a carrier (such as apolymer, e.g. HPMC) and solvent and freeze-drying the mixture (with theintention to provide the compound in amorphous form, rather than incrystalline form), for stability reasons it may be advantageous toincrease the ratio of the amount of carrier to the amount of compound toavoid re-crystallisation of the compound upon standing.

In certain instances, it may be advantageous to administer the compoundof the present invention in combination with at least one additionalpharmaceutical (or therapeutic) agent (e.g., an anti-proliferative oranti-cancer agent or adjunct therapy typically used in chemotherapy).The compound of the present invention may be administered eithersimultaneously with, or before or after, one or more other therapeuticagent(s). Alternatively, the compound of the present invention may beadministered separately, by the same or different route ofadministration, or together in the same pharmaceutical composition asthe other agent(s). Suitable additional anti-cancer agents include butare not limited to,

HER2 and HER3 receptor inhibitors: As recently exemplified in HER2positive breast cancer models, PI3K inhibition will lead to pathwayreactivation, through a FoxO dependent HER2/HER3 transcriptionalinduction, implying the use of HER2 inhibitors in this setting (Serra etal, 2011 Oncogene 30; Chandarlapaty et al, 2011 Cancer Cell 19;Chakrabarty et al 2012, PNAS 109). For example Trastuzumab (sold underthe trademark Herceptin® by Genentech/Roche), pertuzumab (sold under thetrademark Perjeta™, by Genentech/Roche), the antibody-drug conjugateTrastuzumab Emtansine (T-DM1) from Genentech/Roche, erlotinib (soldunder the trademark Tarceva®, by Genentech/Roche, gefitinib (sold underthe trademark Iressa™, by AstraZeneca), MOR10703, neratinib (also knownas HKI-272,(2E)-N-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide,and described in PCT Publication No. WO 05/028443), lapatinib orlapatinib ditosylate (sold under the trademark Tykerb® byGlaxoSmithKline). Such combination being useful in for example HER2positive breast cancers and HER2 amplified gastric cancers. As atherapeutic target, HER3 (ErbB3) presents with the challenge of havingan inactive tyrosine kinase, thus precluding the utility of ATP-mimetictyrosine kinase inhibitors (TKIs). Circumventing this challenge areantibody-mediated strategies aimed at blocking ligand binding to ErbB3(e.g., MM-121) or blocking the dimerization of ErbB3 with ErbB2 inErbB2-overexpressing cells (e.g, pertuzumab)

Estrogen receptor downregulators/aromatase inhibitors: For exampleFulvestrant (sold under the tradename Faslodex®), Letrozole (sold underthe trademark Femara® by Novartis) or Exemestane (sold under thetrademark Aromasin® by Pfizer). Such combination being useful in thetreatment of for example ER positive breast cancer. The rationale forthe combination being aimed to address PI3K related hormone resistance.

Mitogen-activated protein kinase kinase (MEK) inhibitors: For exampleXL-518 (Cas No. 1029872-29-4, available from ACC Corp.), AZD6244 orselumetinib (AstraZeneca), GSK1120212 (GlaxoSmithKline), AZD8330(AstraZeneca), or MEK162. Such combination being useful in the treatmentof for example KRAS mutant lung, colorectal cancer (CRC) and pancreaticcancers.

Bcl2/BclXL inhibitors: for example ABT737 (Abbott).

Anti-androgens: For example Nilutamide (sold under the tradenamesNilandron® and Anandron®), bicalutamide (sold under tradename Casodex®),flutamide (sold under the tradename Fulexin™), MDV3100 (Enzalutamide,sold under the tradename Xtandi® by Medivation) and Abiraterone (soldunder the tradename Zytiga® by Janssen). Such combination being usefulin the treatment of for example hormone dependent prostate cancer withPTEN inactivation. The rationale for the combination being aimed toaddress cross talk between PI3K and Androgen Receptor pathways.

Heat Shock Protein90 (HSP90) inhibitors: For example Tanespimycin(17-allylamino-17-demethoxygeldanamycin, also known as KOS-953 and17-AAG, available from SIGMA, and described in U.S. Pat. No. 4,261,989)and5-(2,4-Dihydroxy-5-isopropyl-phenyl)-4-(4-morpholin-4-ylmethyl-phenyl)-isoxazole-3-carboxylicacid ethylamide (also known as AUY922 and described in PCT PublicationNo. WO2004/072051). Such combination being useful in the treatment offor example EGFR dependent lung cancers, or for inhibiting EGRmut whichbecome refractory to EGR inhibitors, or in HER2 positive breast cancer,or HER2 positive gastric cancer.

Taxane anti-neoplastic agents: For example Cabazitaxel(1-hydroxy-7β,10β-dimethoxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-triyl-4-acetate-2-benzoate-13-[(2R,3S)-3-{[(tert-butoxy)carbonyl]amino}-2-hydroxy-3-phenylpropanoate),larotaxel((2α,3ξ,4α,5β,7α,10β,13α)-4,10-bis(acetyloxy)-13-({(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropanoyl}oxy)-1-hydroxy-9-oxo-5,20-epoxy-7,19-cyclotax-11-en-2-ylbenzoate);

Anti-mitotic agents: For example Docetaxel (sold under the tradenameTaxotere® by Sanofi-Aventis), useful for the treatment of breast cancer.

Plant Alkaloids: For example paclitaxel (sold under the tradenames Taxoland Onxal™) and Paclitaxel protein-bound (sold under the tradenameAbraxane®) and useful for the treatment of prostate cancer, vinblastine(also known as vinblastine sulfate, vincaleukoblastine and VLB, soldunder the tradenames Alkaban-AQ® and Velban®), vincristine (also knownas vincristine sulfate, LCR, and VCR, sold under the tradenames Oncovin®and Vincasar Pfs®) and vinorelbine (sold under the tradenameNavelbine®).

Anti-Insulin-like Growth Factor-1 receptor (IGF-1R) antibodies: Forexample Figitumumab (also known as CP-751,871, available from ACC Corp),and robatumumab (CAS No. 934235-44-6).

PARP (poly ADP-ribose polymerase) inhibitors: For example BSI-201(iniparib) and olaparib. Such combination being useful in for exampleaddressing possible induction of DNA damage machinery by PI3Kinhibitors.

Suitable therapeutic agents for adjunct therapy include steroids,anti-inflammatory agents, anti-histamines, antiemetics, and other agentswell-known to those of skill in art for use in improving the quality ofcare for patients being treated for the diseases, conditions, ordisorders described herein.

Because activation of the PI3K/Akt pathway drives cell survival,inhibition of the pathway in combination with therapies that driveapoptosis in cancer cells, including radiotherapy and chemotherapy, mayresult in improved responses (Ghobrial et al., CA Cancer J. Clin55:178-194 (2005)). As an example, combination of PI3 kinase inhibitorwith carboplatin demonstrated synergistic effects in both in vitroproliferation and apoptosis assays as well as in in vivo tumor efficacyin a xenograft model of ovarian cancer (Westfall and Skinner, Mol.Cancer. Ther. 4:1764-1771 (2005)). Compounds of the present inventionmay be administered in conjunction with radiotherapy.

The compound of the present invention or pharmaceutical compositionthereof can be administered by the following routes: enteral, such asnasal; rectal or oral; parenteral, such as intramuscular or intravenous;or topical, such as dermal administration. The compound of the presentinvention or pharmaceutical composition thereof for use in humans ispreferably administered orally (e.g. in tablet form).

The pharmaceutical composition or combination of the present inventioncan be in unit dosage of about 1 mg to about 1000 mg of activeingredient(s) for a subject of about 50 kg to about 70 kg, or about 1 mgto about 500 mg or about 1 mg to about 250 mg or about 1 mg to about 150mg or about 0.5 mg to about 100 mg, or about 1 mg to about 50 mg ofactive ingredients. Unit dosage can also be of about 50 mg to about 1000mg of active ingredient(s) for a subject of about 50 kg to about 70 kg,or about 50 mg to about 500 mg or about 50 mg to about 250 mg or about50 mg to about 150 mg or about 50 mg to about 100 mg of activeingredients. Unit dosage can also be of about 100 mg to about 500 mg ofactive ingredient(s) for a subject of about 50 kg to about 70 kg, orabout 200 mg to about 500 mg or about 300 mg to about 500 mg or about300 mg to about 400 mg of active ingredients. These dosages may beprovided as the total daily dosage, and may be provided in unit dosageor in split dosages. The dosage may depend upon the particular dosageform used for delivering the active ingredient(s). In general, thetherapeutically effective dosage of a compound, the pharmaceuticalcomposition, or the combinations thereof, is dependent on the species ofthe subject, the body weight, age and individual condition, the disorderor disease or the severity thereof being treated. The dosage can alsodepend on the bioavailability of the active ingredient in the speciesbeing treated. A physician, pharmacist, clinician or veterinarian ofordinary skill can readily determine the effective amount of each of theactive ingredients necessary to prevent, treat or inhibit the progressof the disorder or disease.

The above-cited dosage properties are demonstrable in vitro and in vivotests using advantageously mammals, e.g., mice, rats, dogs, monkeys orisolated organs, tissues and preparations thereof. The compounds of thepresent invention can be applied in vitro in the form of solutions,e.g., aqueous solutions prepared from e.g. 10 mM DMSO stock solution,and in vivo either enterally, parenterally, advantageouslyintravenously, e.g., as a suspension or in aqueous solution. The dosagein vitro may range between about 10⁻³ molar and about 10⁻⁹ molarconcentrations. A therapeutically effective amount in vivo may rangedepending on the route of administration, between about 0.1 to about 500mg/kg, or between about 1 to about 100 mg/kg.

In general, a therapeutically effective amount of a compound of thepresent invention is administered to a patient in need of treatment. Theterm “a therapeutically effective amount” of a compound of the presentinvention refers to an amount of the compound of the present inventionthat will elicit the biological or medical response of a subject, forexample, reduction or inhibition of an enzyme activity or a proteinactivity or a protein complex activity, or ameliorate symptoms,alleviate conditions, slow or delay disease progression, or prevent adisease, etc.

In yet another aspect, a method for treating cancer in a mammal isprovided which comprises administering to a mammal in need of suchtreatment an effective amount of a compound of the present invention.

As used herein, the term “subject” refers to an animal. Typically theanimal is a mammal. A subject also refers to for example, primates(e.g., humans, male or female), cows, sheep, goats, horses, dogs, cats,rabbits, rats, mice, fish, birds and the like. In certain embodiments,the subject is a primate. Preferably, the subject is a human.

As used herein, the term “inhibit”, “inhibition” or “inhibiting” refersto the reduction or suppression of a given condition, symptom, ordisorder, or disease, or a significant decrease in the baseline activityof a biological activity or process.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder, refers (i) to ameliorating the disease or disorder(i.e., slowing or arresting or reducing the development of the diseaseor at least one of the clinical symptoms thereof); (ii) to alleviatingor ameliorating at least one physical parameter including those whichmay not be discernible by the patient; or (iii) to preventing ordelaying the onset or development or progression of the disease ordisorder. In general, the term “treating” or “treatment” describes themanagement and care of a patient for the purpose of combating thedisease, condition, or disorder and includes the administration of acompound of the present invention to prevent the onset of the symptomsor complications, alleviating the symptoms or complications, oreliminating the disease, condition or disorder.

As used herein, a subject is “in need of” a treatment if such subjectwould benefit biologically, medically or in quality of life from suchtreatment (preferably, the subject is a human).

Another aspect of the invention is a product comprising a compound ofthe present invention and at least one other therapeutic agent (orpharmaceutical agent) as a combined preparation for simultaneous,separate or sequential use in therapy to enhance apoptosis.

In the combination therapies of the invention, the compound of thepresent invention and the other therapeutic agent may be manufacturedand/or formulated by the same or different manufacturers. Moreover, thecompound of the present invention and the other therapeutic (orpharmaceutical agent) may be brought together into a combinationtherapy: (i) prior to release of the combination product to physicians(e.g. in the case of a kit comprising the compound of the invention andthe other therapeutic agent); (ii) by the physician themselves (or underthe guidance of the physician) shortly before administration; (iii) inthe patient themselves, e.g. during sequential administration of thecompound of the invention and the other therapeutic agent.

Accordingly, the invention provides the use of a compound of the presentinvention for treating a disease or condition by inhibiting orantagonizing PI3K, wherein the medicament is prepared for administrationwith another therapeutic agent. The invention also provides for the useof another therapeutic agent, wherein the medicament is administered asa combination of a compound of the present invention with the othertherapeutic agent.

Embodiments of the present invention are illustrated by the followingExamples. It is to be understood, however, that the embodiments of theinvention are not limited to the specific details of these Examples, asother variations thereof will be known, or apparent in light of theinstant disclosure, to one of ordinary skill in the art.

EXAMPLES

Unless specified otherwise, starting materials are generally availablefrom commercial sources such as Aldrich Chemicals Co. (Milwaukee, Wis.),Lancaster Synthesis, Inc. (Windham, N.H.), Acros Organics (Fairlawn,N.J.), Maybridge Chemical Company, Ltd. (Cornwall, England), TygerScientific (Princeton, N.J.), Chem-Impex International, Inc. (Wood Dale,Ill.), and AstraZeneca Pharmaceuticals (London, England).

The abbreviations used in the following Examples have the correspondingmeanings listed below.

-   -   AcOH acetic acid    -   AlCl₃ aluminium trichloride    -   API atmospheric pressure ionization    -   Boc tert-butoxycarbonyl    -   Brine saturated (at rt) sodium chloride solution    -   br. s broad singulet    -   ^(n)BuOH n-butanol    -   ^(t)Bu tert-butyl    -   CDI carbonyl diimidazole    -   Celite trademark of Celite Corp. (World Minerals Inc.), Santa        Barbara, Calif., USA, for filtering aid based on kieselguhr    -   CH₃CN acetonitrile    -   conc. concentrated    -   d doublett    -   DCE dichloroethane    -   DCM dichloromethane    -   DEA diethylamine    -   DIEA N,N-diethyl-isopropylamine    -   DMAP 4-dimethylaminopyridine    -   DME dimethoxyethane    -   DMF N,N-dimethylformamide    -   DMSO dimethylsulfoxide    -   ES-MS electrospray mass spectrometry    -   Et ethyl    -   Et₃N triethylamine    -   Et₂O diethyl ether    -   EtOAc ethyl acetate    -   EtOH ethanol    -   H hour(s)    -   HPLC high-performance liquid chromatography    -   Hyflo Hyflo Super Cel®    -   iPr isopropyl    -   K₂CO₃ potassium carbonate    -   KOH potassium hydroxide    -   K₃PO₄ potassium phosphate    -   LAH lithium aluminium hydride    -   LC liquid chromatography    -   Me methyl    -   MeI methyl iodide    -   MeOH methanol    -   MgSO₄ magnesium sulfate    -   M multiplett    -   min minute(s)    -   mL milliliter(s)    -   m.p. melting point    -   MS Mass Spectrometry    -   NaH sodium hydride    -   NaHCO₃ sodium bicarbonate    -   Na₂CO₃ sodium carbonate    -   NaHMDS sodium hexamethyldisilazane    -   NaOH sodium hydroxide    -   Na₂SO₄ sodium sulfate    -   MgSO₄ magnesium sulfate    -   NaOAc sodium acetate    -   NBS N-bromosuccinimide    -   NH₄Cl ammonium chloride    -   NH₄OH ammonium hydroxide    -   NMR nuclear magnetic resonance    -   POCl₃ phosphorus (III) oxychloride    -   RT room temperature    -   R_(f) TLC retention factor    -   S singulet    -   scCO₂ super critical CO₂    -   t triplet    -   TBAF tetrabutylammonium fluoride    -   TBDPSCl tert-Butyldiphenylsilyl chloride    -   TBME tert-butylmethylether    -   TEA triethylamine    -   TEMPO 2,2,6,6-tetramethylpiperidinyloxyl    -   TFA trifluoroacetic acid    -   THF tetrahydrofurane    -   TLC thin layer chromatography    -   TMS trimethylsilyl    -   TMSCl trimethylsilyl chloride    -   t_(R) time of retention    -   TsCl p-toluenesulfonyl chloride    -   TsOH p-toluenesulfonic acid    -   UV ultraviolet        General Method

1H-NMR measurements were performed on a Bruker Ultrashield™ 400 (400MHz), Bruker Ultrashield™ 600 (600 MHz) or a 500 MHz DRX BrukerCryoProbe (500 MHz) spectrometer using or not trimethylsilane as aninternal standard. Chemical shifts (d-values) are reported in ppmdownfield from tetramethylsilane, coupling constants (J) are given inHz, spectra splitting pattern are designated as singulet (s), doublet(d), doublet doublet (dd), triplet (t), quadruplet (q), multiplet ormore overlapping signals (m), broad signal (br). Solvents are given inparentheses.

TLC were performed with precoated silica gel 60 F₂₅₄ glass plates(Merck, Darmstadt, Germany) using the respective named solvent systems.Visualization was generally done by UV light (254 nm).

HPLC Conditions:

LC-MS 1:

Column: Acquity HSS T3 2.1×50 mm, 1.8 μm. Flow: 1.2 mL/min. Columntemperature: 50° C.

Gradient: 2% to 98% B in 1.4 min, 98% B for 0.75 min, 98% to 2% B in0.04 min, 2% B for 0.01 min; A=water+0.05% formic acid+3.75 mM ammoniumacetate, B=acetonitrile+0.04% formic acid

Detection full scan: 215-350 nm

LC-MS 2:

Column: Acquity HSS T3 2.1×50 mm, 1.8 μm. Flow: 1.2 mL/min. Columntemperature: 50° C.

Gradient: 2% to 98% B in 1.4 min, 98% B for 0.75 min, 98% to 2% B in0.04 min, 2% B for 0.01 min; A=water+0.05% formic acid+0.05% ammoniumacetate, B=acetonitrile+0.04% formic acid

Detection full scan: 215-350 nm

LC-MS 3:

Column: Acquity HSS T3 2.1×50 mm, 1.8 μm. Flow: 1.0 mL/min. Columntemperature: 60° C.

Gradient: 5% to 98% B in 1.4 min, 98% B for 0.75 min, 98% to 5% B in0.04 min, 5% B for 0.01 min; A=water+0.05% formic acid+3.75 mM ammoniumacetate, B=acetonitrile+0.04% formic acid

Detection full scan: 215-350 nm

HPLC 1:

Column: Chromolith performance RP18e 4.6×100 mm, Flow: 2.0 mL/min.Gradient: 2% to 100% B in 4.5 min, 100% B for 1 min, A=water+0.1% TFA,B=acetonitrile+0.1% TFA

Detection: 215 nm

UPLC 1:

Column: Acquity UPLC HSS T3 C18, 1.7 μm 2.1×50 mm, Flow: 1.0 mL/min.Gradient: 5% to 100% B in 1.5 min, 100% B for 1 min, A=water+0.1% TFA,B=acetonitrile+0.1% TFA

Detection: 218 nm

Intermediate A: 4-(4,6-Dichloro-pyrimidin-2-yl)-morpholine

Intermediate A is commercially available or can be prepared using thefollowing procedure.

To a solution of 2,4,6-trichloropyrimidine (5.0 mL, 42.6 mmol) inmesitylen (80 mL) at 165° C. was added dropwise a solution of morpholine(4.83 mL, 55.4 mmol) in mesitylen (20 mL) and the suspension was stirredat 165° C. for 30 min. The reaction mixture was treated with H₂O, EtOAcand NaHCO₃. The organic layer was washed with H₂O and brine, dried(Na₂SO₄), filtered and concentrated. The residue was purified by flashchromatography (hexane/EtOAc, 100:0→7:3). The residue was triturated inhexane and filtered to afford the title compound (3.36 g, 33%). t_(R):1.11 min (LC-MS 1); ESI-MS: 234.2 [M+H]⁺ (LC-MS 1).

Intermediate B:5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-4-trifluoromethyl-pyrimidin-2-ylamine

To a suspension of the product from step B.1 (16.2 g, 66.3 mmol),bis-pinacolatodiboron (18.5 g, 72.9 mmol) and KOAc (19.5 g, 199 mmol) indioxane (300 mL) under argon was added PdCl₂(dppf)CH₂Cl₂ adduct (2.4 g,2.98 mmol) and the mixture was stirred at 115° C. for 4 h. The reactionmixture was cooled to 50° C. and treated with EtOAc. The resultingsuspension was filtered over Hyflo and washed with EtOAc. The combinedfiltrated were concentrated. The residue was suspended in 2 N NaOH,stirred at RT for 5 min and then Et₂O and H₂O were added and the binarymixture was filtered through Hyflo. The phases of the filtrate wereseparated. The pH of the resulting aqueous layer was adjusted to 5-6with HCl 4N and then extracted with EtOAc. The organic layer was washedwith H₂O and brine, dried (Na₂SO₄), filtered and concentrated. Theresidue was triturated in Et₂O and hexane, filtered to afford the titlecompound (8.33 g, 42%). t_(R): 1.00 min (LC-MS 1); ESI-MS: 290.3 [M+H]⁺(LC-MS 1).

Step B1: 5-Bromo-4-trifluoromethyl-pyrimidin-2-ylamine

To a solution of 2-amino-4-trifluoromethylpyrimidine (25 g, 0.15 mol) inCH₃CN (800 mL) was added dropwise (over 2.5 hours) NBS (34.8 g, 0.195mol) dissolved in 200 mL of CH₃CN in the dark. The mixture was stirred4.5 h at RT in the dark and then the solvent was evaporated. The residuewas dissolved in EtOAc and H2O and the binary mixture was transferredinto a separating funnel. The aqueous layer was separated and extractedwith EtOAc. The organic layers were washed with H2O and brine, driedwith Na2SO4, filtered and evaporated. The residue was purified bychromatography on silica gel using a gradient of hexane/EtOAc 9:1 to3:2. The combined pure fractions were evaporated and the residuesuspended in 40 mL hexane, stirred for 10 min., filtered and washed with2×20 mL of hexane to give the title product as a beige solid (31.2 g,85%). t_(R): 0.82 min (LC-MS 1).

Example 1(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-methyl-oxazolidin-2-one

A solution of product from step 1.1 (350 mg, 1.16 mmol), intermediate B(449 mg, 1.51 mmol), Na₂CO₃(2M, 1.7 mL, 3.48 mmol) andPdCl₂(dppf)-CH₂Cl₂(95 mg, 0.17 mmol) in DME (10 mL) under argon wasstirred at 80° C. for 1 h. The mixture was diluted in EtOAc andextracted with saturated NaHCO₃. The organic layer was washed with H₂Oand brine, dried (Na₂SO₄), filtered and concentrated. The residue waspurified by flash chromatography (CH₂Cl₂/EtOH, 99.5:0.5→98:2). Theresidue was triturated in hexane, filtered and dried. The residue waspurified by preparative HPLC (Waters Sun Fire C18, 30×100 mm, 5 um; 0.1%TFA-water/acetonitrile; gradient acetonitrile 5-100% in 20 min) toafford the title compound (260 mg, 52%). t_(R): 0.93 min (LC-MS 1);ESI-MS: 426.3 [M+H]⁺ (LC-MS 1).

Step 1.1:(S)-3-(6-Chloro-2-morpholin-4-yl-pyrimidin-4-yl)-4-methyl-oxazolidin-2-one

To a solution of (S)-4-methyl-2-oxazolidinone (432 mg, 4.19 mmol) in DMF(10 mL) was slowly added NaH (60% mineral oil, 201 mg, 5.02 mmol) underan argon atmosphere and the suspension was stirred at rt for 30 min. Thereaction mixture was cooled to 0° C. and the intermediate A (1 g, 4.19mmol) was added. The mixture was stirred at RT for 4 h. The reactionmixture was diluted with EtOAc and extracted with H₂O. The organic layerwas washed with H₂O and brine, dried (Na₂SO₄), filtered andconcentrated. The residue was purified by flash chromatography(hexane/EtOAc, 97:3→1:1) to afford the title compound (605 mg, 47%).t_(R): 1.00 min (LC-MS 1); ESI-MS: 299.2/301.2 [M+H]⁺ (LC-MS 1).

Example 2(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5,5-dimethyl-oxazolidin-2-one

A solution of the product from step 2.1 (28 mg, 0.04 mmol) and TBAF (2mL, 2.0 mmol, 1M in THF) was stirred at rt overnight. The reactionmixture was concentrated and the residue was purified by flashchromatography (DCM/MeOH, 100:0→95:5) to give the title product. t_(R):0.89 min (LC-MS 1); ESI-MS: 470.2 [M+H]⁺ (LC-MS 1).

Step 2.1:(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-(tert-butyl-diphenyl-silanyloxymethyl)-5,5-dimethyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure used forexample 1 but using the product from step 2.2. The mixture was performedat 100° C. for 40 min. After extraction, the residue was purified byflash chromatography (heptane/EtOAc, 100:0→30:70) to give the titleproduct. t_(R): 1.45 min (LC-MS 1); ESI-MS: 708.4 [M+H]⁺ (LC-MS 1).

Step 2.2:(S)-4-(tert-Butyl-diphenyl-silanyloxymethyl)-3-(6-chloro-2-morpholin-4-yl-pyrimidin-4-yl)-5,5-dimethyl-oxazolidin-2-one

A solution of the product from step 2.3 (95 mg, 0.25 mmol), intermediateA (58 mg, 0.25 mmol), xantphos (10 mg, 0.02 mmol), Pd₂ dba₃ (4.5 mg,4.95 umol) and Cs₂CO₃ (121 mg, 0.37 mmol) in dioxane under argon wasstirred at 100° C. for 3 h. The mixture was cooled to rt, diluted withEtOAc and extracted with a saturated NaHCO₃ solution. The organic layerwas washed with brine, dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by flash chromatography (heptane/EtOAc,100:0→0:100) to give the title product (85 mg, 56%). t_(R): 1.54 min(LC-MS 1); ESI-MS: 581.4/583.3 [M+H]⁺ (LC-MS 1).

Step 2.3:(S)-4-(tert-Butyl-diphenyl-silanyloxymethyl)-5,5-dimethyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure used forstep 6.2 but using the product from step 2.4, and using Et₃N instead ofimidazole. The mixture was stirred at RT for 16 h. The reaction mixturewas concentrated and purified by flash chromatography (heptane/EtOAc,100:0→55:45) to give the title product. t_(R): 1.33 min (LC-MS 1);ESI-MS: 384.3 [M+H]⁺ (LC-MS 1).

Step 2.4: (S)-4-Hydroxymethyl-5,5-dimethyl-oxazolidin-2-one

A solution of the product from step 2.5 (110 mg, 0.59 mmol) and HCl (4Min dioxane, 5 mL, 20 mmol) was stirred at RT for 4 h. The reactionmixture was concentrated and the residue was used without furtherpurification.

Step 2.5: (S)-1,1,5,5-Tetramethyl-dihydro-oxazolo[3,4-c]oxazol-3-one

To a solution of the product from step 2.6 (190 mg, 0.73 mmol) in DMF (6mL) under argon at 0° C. was added NaH (88 mg, 2.20 mmol, 60% in oil)and the mixture was stirred at 0° C. for 6 h. The reaction mixture wasquenched with H₂O and concentrated. The residue was triturated in EtOAcand filtered. The filtered solution was dried (Na₂SO₄), filtered andconcentrated. The product was purified by flash chromatography(heptane/EtOAc, 100:0→60:40).

Step 2.6:(S)-4-(1-Hydroxy-1-methyl-ethyl)-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester

To a solution of (S)-3-tert-butyl 4-methyl2,2-dimethyloxazolidine-3,4-dicarboxylate (500 mg, 1.93 mmol) in THF (15mL) under argon at 0° C. was added dropwise methylmagnesium bromide (1.4mL, 4.24 mmol) and the mixture was stirred at 0° C. for 2 h. Thereaction was quenched with a saturated NH₄Cl solution and extracted withEtOAc. The organic layer was washed with brine, dried (Na₂SO₄), filteredand concentrated. The residue was purified by flash chromatography(heptane/EtOAc, 100:0→65:35). t_(R): 1.01 min (LC-MS 1); ESI-MS: 260.3[M+H]⁺ (LC-MS 1).

Example 3 Racemic3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-4,5′-bipyrimidin-6-yl)-4-(hydroxymethyl)-4-methyloxazolidin-2-one

A solution of intermediate B (68 mg, 0.21 mmol), the product from step3.1 (120 mg, 021 mmol), a 2M Na₂CO₃ solution (317 uL, 0.63 mmol) andtetrakis (15 mg, 0.01 mmol) in DME (2 mL) was stirred at 80° C. for 3 h.The reaction mixture was diluted in EtOAc and Na₂SO₄ was added. Theresulting suspension was filtered and the filtrate was concentrated. Theresidue was dissolved with THF (2 ML) and TBAF (212 uL, 0.21 mmol) wasadded. The mixture was stirred at rt for 16 h and was concentrated. Thecrude was purified by flash chromatography (DCM/EtOH, 99:1→96:4). Theresidue was triturated in DCM/hexane to afford the title compound.t_(R): 0.85 min (LC-MS 1); ESI-MS: 456.3 [M+H]⁺ (LC-MS 1).

Step 3.1:4-(tert-Butyl-diphenyl-silanyloxymethyl)-3-(6-chloro-2-morpholin-4-yl-pyrimidin-4-yl)-4-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 2.2 but using product from step 3.2. After extraction, theresidue was purified by flash chromatography (hexane/EtOAc: 9:1→1:1) togive the title compound.

Step 3.2:4-(tert-Butyl-diphenyl-silanyloxymethyl)-4-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 6.4 but using 4-(hydroxymethyl)-4-methyloxazolidin-2-one, andusing DCM instead of DMF. The reaction mixture was extracted with Et₂O.The organic layer was washed with H₂O and brine, dried (Na₂SO₄),filtered and concentrated. The resulting solid was triturated in hexaneand filtered to afford the title compound. t_(R): 1.20 min (LC-MS 1);ESI-MS: 339.2/341.2 [M+H]⁺ (LC-MS 1).

Example 3A First eluting enantiomer of3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-4,5′-bipyrimidin-6-yl)-4-(hydroxymethyl)-4-methyloxazolidin-2-one

Absolute stereochemistry not determined.

The title compound was obtained after preparative chiral SFC separationof the racemic product of example 3. (Column: Chiralpak AD-H, 30×250 mm.Flow 80 mL/min. scCO₂/MeOH 85:15). t_(R): 3.97 min (Column: ChiralpakAD-H, 4.6×250 mm. Flow 3 mL/min. scCO₂/MeOH 85:15).

Example 3B Second eluting enantiomer of3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-4,5′-bipyrimidin-6-yl)-4-(hydroxymethyl)-4-methyloxazolidin-2-one

Absolute stereochemistry not determined.

The title compound was obtained after preparative chiral SFC separationof the racemic product of example 3. (Column: Chiralpak AD-H, 30×250 mm.Flow 80 mL/min. scCO₂/MeOH 85:15). t_(R): 4.49 min (Column: ChiralpakAD-H, 4.6×250 mm. Flow 3 mL/min. scCO₂/MeOH 85:15).

Example 4(3aS,7aS)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-hexahydro-benzooxazol-2-one

A solution of product from step 4.1 (60 mg, 0.17 mmol), intermediate B(54 mg, 0.17 mmol), Na₂CO₃(2M, 260 μL, 0.52 mmol) and palladium tetrakis(10 mg, 8.7 μmol) in DME (1.5 mL) under argon was stirred at 80° C. for2 h in a sealed vial. The reaction mixture was concentrated. The residuewas purified by flash chromatography (CH₂Cl₂/EtOH, 99.8:0.2→97.5:2.5).The residue was dissolved in DCM (2 mL) and then treated in hexane (4mL). The crystals were filtered and washed with hexane (3 ml) to givethe title compound (36 mg, 44%). t_(R): 1.10 min (LC-MS 1); ESI-MS:466.3 [M+H]⁺ (LC-MS 1).

Step 4.1:(3aS,7aS)-3-(6-Chloro-2-morpholin-4-yl-pyrimidin-4-yl)-hexahydro-benzooxazol-2-one

The title compound was prepared in analogy to the procedure describedfor step 2.2, but using product from step 4.2. The reaction wasperformed at 100° C. for 1 h. The reaction mixture was filtered throughHyflo and concentrated. The residue was purified by flash chromatography(hexane/EtOAc: 9:1→1:1) to give the title compound. t_(R): 1.20 min(LC-MS 1); ESI-MS: 339.2/341.2 [M+H]⁺ (LC-MS 1).

Step 4.2: (3aS,7aS)-Hexahydro-benzooxazol-2-one

The (1S,2S)-2-Aminocyclohexanole (750 mg, 6.51 mmol) and the2-Nitrophenyl chloroformate (1378 mg, 6.84 mmol) were stirred in DCE (15mL) with DIEA (2.39 mL, 13.68 mmol) in a sealed vial at 90° C. for 1 h.The reaction mixture was given into a separating funnel with 50 mL EtOAcand 50 mL saturated NaHCO₃ solution. The aqueous layer was washed with50 mL EtOAc. The organic layers were combined and washed with 50 mL H2O,50 mL brine, dried with Na₂SO₄, filtered and concentrated. The residuewas purified by flash chromatography (hexane/EtOAc: 7:3→3:7) to give thetitle compound (770 mg, 5.13 mmol). t_(R): 0.69 min (LC-MS 1); ¹H NMR(400 MHz, <dmso>) δ ppm 1.19-1.44 (m, 3H) 1.45-1.61 (m, 1H) 1.65 (d,J=9.77 Hz, 1H) 1.76 (d, J=11.34 Hz, 1H) 1.82-1.93 (m, 1H) 1.93-2.10 (m,1H) 3.03-3.23 (m, 1H) 3.74 (td, J=11.34, 3.52 Hz, 1H) 7.53 (br. s., 1H)

Example 5(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-methoxymethyl-oxazolidin-2-one

In a microwave vial, to a solution of product from step 5.1 (116 mg,0.28 mmol) and intermediate B (90 mg, 0.31 mmol) in DME (2.1 mL) wereadded saturated Na₂CO₃ solution (0.7 ml) and PdCl₂(dppf)₂.CH₂Cl₂ (23 mg,0.03 mmol). The mixture was bubbled with argon for 5 min. It was stirredat 120° C. for 15 min under microwave irradiations. The reaction mixturewas taken up in DCM and water. Layers were separated and aqueous layerwas extracted twice more with some DCM. Then organic layers werecombined, dried over sodium sulfate and evaporated. The residue waspurified by flash chromatography (DCM/MeOH: 100%→95% DCM). The residueobtained was purified by reverse phase flash chromatography (MeCN/H₂O:10%→100% MeCN) to give the title compound (19 mg, 13%). t_(R): 0.91 min(LC-MS 1); ESI-MS: 456.1 [M+H]⁺ (LC-MS 1).

Step 5.1:(S)-3-(6-Chloro-2-morpholin-4-yl-pyrimidin-4-yl)-4-methoxymethyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 2.2, but using product from step 5.2. The reaction wasperformed at 115° C. for 80 min. The reaction mixture was concentratedand taken up with DCM/water. The layers were separated and the aqueousone was extracted three times with DCM. The organic layers were combinedand dried over sodium sulfate. The residue was purified by flashchromatography (heptane/EtOAc: 100%→60% heptane.) to give the titlecompound (116 mg, 22%). t_(R): 0.98 min (LC-MS 1); ESI-MS: 329.2 [M+H]⁺(LC-MS 1).

Step 5.2: (S)-4-Methoxymethyl-oxazolidin-2-one

TsOH (800 mg, 4.21 mmol) was added to a yellow solution of product fromstep 5.3 (1.013 g, 4.13 mmol) in MeOH (10 ml). The mixture was stirredat RT for 90 min. Then TsOH (140 mg, 0.74 mmol) was added and it wasstirred for 70 min at RT. Then solvent was removed and the residue wasdissolved in DCM (6 mL) with Triethylamine (1.44 ml, 10.32 mmol). Asolution of triphosgene (0.613 g, 2.07 mmol) in DCM (4 mL) was slowlyadded to the mixture. The reaction mixture was stirred at RT for 2 h 30.The reaction was quenched with a few drops of water. It was thenacidified to pH=4 with buffer was added and then the layers wereseparated. The aqueous one was extracted once more with some DCM.Organic layers were combined, dried over sodium sulfate and evaporated.The residue was purified by flash chromatography (DCM/MeOH: 100%→90%DCM) to give the title compound (231 mg, 38%). ESI-MS: 132.1 [M+H]⁺(LC-MS 1).

Step 5.3: (S)-4-Methoxymethyl-2,2-dimethyl-oxazolidine-3-carboxylic acidtert-butyl ester

NaH (265 mg, 6.63 mmol) was added to a yellow solution of(S)-1-Boc-2,2-dimethyl-4-hydroxymethyloxazolidine (AstaTech Inc.,Bristol, Pa.) (1 g, 4.19 mmol) in THF (10 ml). Then the mixture wasstirred for 15 min at ambient temperature. The Methyliodid (323 μL, 5.19mmol) was added to the yellow suspension and the mixture was stirred for2 h 30 at rt. Then water was added to quench the reaction. The solventwas removed. The residue was purified by flash chromatography (DCM/MeOH:5%→10% MeOH) to give the title compound (1.013 g, 94%). ESI-MS: 246.1[M+H]⁺ (LC-MS 1); ¹H NMR (400 MHz, <cdcl3>) δ ppm 1.48 (s, 9H) 1.53 (br.s., 6H) 3.30 (m, 1H) 3.36 (s, 3H) 3.41-3.63 (m, 2H) 3.88-4.00 (m, 2H)

Example 6(4S,5S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-O-4-hydroxymethyl-5-methyl-oxazolidin-2-one

A solution of product from step 6.1 (600 mg, 0.82 mmol) in THF (5 mL)was treated with HF.pyridine in THF (7.14 mL, 57.5 mmol) for 4 days atRT in a plastic vial. Then the reaction mixture was added dropwise to astirred a mixture of saturated NaHCO₃ solution (300 mL) and EtOAc (200mL). Then solid NaHCO3 was added until pH˜8 and the layers wereseparated. The aqueous one was washed with 100 mL EtOAc, The organicextracts were combined and washed with water and brine. It was thendried over Na₂SO₄, filtered and evaporated. The crude was purified byflash chromatography (DCM/EtOH: 99:1→95:5). The fractions were combinedand concentrated. The residue was sonicated in DCM and then hexane wasadded. The crystals obtained were filtered off and re-purified 3 timesby flash chromatography (DCM/EtAOc: 9:1→3:7, then Hexane/THF: 9:1→1:1,then Hexane/THF: 7:3→1:1) to give the title compound (243 mg, 64%).t_(R): 0.80 min (LC-MS 1); ESI-MS: 456.6 [M+H]⁺ (LC-MS 1).

Step 6.1:(4S,5S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-(tert-butyl-diphenyl-silanyloxymethyl)-5-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor example 1, but using product from step 6.2. The reaction wasperformed at 80° C. for 1 h. After extraction, the residue was purifiedby flash chromatography (DCM/EtOH: 95.5:0.5→97:3) to give the titlecompound. t_(R): 1.43 min (LC-MS 1); ESI-MS: 694.5 [M+H]⁺ (LC-MS 1).

Step 6.2:(4S,5S)-4-(tert-Butyl-diphenyl-silanyloxymethyl)-3-(6-chloro-2-morpholin-4-yl-pyrimidin-4-yl)-5-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 2.2, but using product from step 6.3. The reaction wasperformed at 100° C. for 3 h 30. The reaction mixture was taken up withEtOAc and washed with saturated NaHCO₃ solution and brine. The organiclayer was and dried over sodium sulfate. The residue was purified byflash chromatography (heptane/EtOAc: 100%→30% heptane.) to give thetitle compound (116 mg, 22%). t_(R): 1.51 min (LC-MS 1); ESI-MS:567.4/569.5 [M+H]⁺ (LC-MS 1).

Step 6.3:(4S,5S)-4-(tert-Butyl-diphenyl-silanyloxymethyl)-5-methyl-oxazolidin-2-one

The product from step 6.4 (3.2 g, 9.31 mmol) was dissolved in DCM (32ml) and treated with Et3N (3.25 ml, 23.29 mmol). The solution wasflushed with Argon and stirred for 5 min at RT. Then, it was treatedwith Triphosgene (1.382 g, 4.66 mmol) and stirred at RT for 16H. Thereaction was quenched with saturated NH4Cl solution (10 mL) and stirred10 min at RT. The water layer was separated and the organic layer washedwith water. The combined aqueous layer were extracted 3× with DCM. Thecombined organic layer were dried over Na₂SO₄, filtered andconcentrated. The residue was purified by flash chromatography(heptane/EtOAc: 100%→50% heptane.) to give the title compound (2.22 g,61%). t_(R): 1.28 min (LC-MS 1); ESI-MS: 387.3 [M+18]⁺ (LC-MS 1).

Step 6.4: (2S,3S)-3-Amino-4-(tert-butyl-diphenyl-silanyloxy)-butan-2-ol

D-threoninol (2 g, 19.02 mmol) was dissolved in DMF (15 ml), treatedwith imidazole (3.89 g, 57.1 mmol) and stirred at RT for 5 min. Then,TBDPS-Cl (5.13 ml, 19.97 mmol) was added dropwise to the reactionsolution under argon. The reaction solution was stirred for 16H at RT.Then it was diluted in EtOAc and washed twice with saturated NaHCO₃solution and once with brine. Organic layer was dried over Na₂SO₄,filtered and concentrated. The residue was purified by flashchromatography (heptane/EtOAc: 100%→0% heptane.) to give the titlecompound (3.21 g, 47%). t_(R): 0.98 min (LC-MS 1); ESI-MS: 344.3 [M+H]⁺(LC-MS 1).

Example 7(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-oxazolidin-2-one

A solution of product from step 7.1 (200 mg, 0.35 mmol), intermediate B(131 mg, 0.39 mmol), Na₂CO₃(2M, 526 μL, 1.05 mmol) and palladiumtetrakis (24 mg, 0.21 mmol) in DME (4 mL) under argon was stirred at 80°C. for 2 h. The reaction mixture was treated with Na₂SO₄, diluted inEtOAc and the insoluble parts were filtered off. The filter cake waswashed three times with EtOAc and the filtrate was evaporated. Then theresidue was dissolved in THF and TBAF solution (1N, 351 μL, 0.35 mmol)was added. The mixture was stirred 1 h at RT. The solvent was removedand the residue was purified by flash chromatography (DCM/EtOH:99:1→95:5) to give the title compound. t_(R): 0.74 min (LC-MS 1).

Step 7.1:(R)-4-(tert-Butyl-diphenyl-silanyloxymethyl)-3-(6-chloro-2-morpholin-4-yl-pyrimidin-4-yl)-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 2.2, but using product from step 7.2. The reaction wasperformed at 100° C. for 3 h. The reaction mixture was filtered and thefiltrate was concentrated. The residue was purified by flashchromatography (hexane/EtOAc: 9:1→6:4) to give the title compound.t_(R): 1.53 min (LC-MS 1); ESI-MS: 553.4/555.5 [M+H]⁺ (LC-MS 1).

Step 7.2: (R)-4-(tert-Butyl-diphenyl-silanyloxymethyl)-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 6.4, but using (S)-4-(hydroxymethyl)oxazolidin-2-one(SpeedChemical Corp. Shanghai), and DCM instead of DMF. The reaction wasperformed at RT for 16 h. The reaction mixture was diluted with waterand extracted twice with Et₂O. The organic layers were combined, washedwith water and brine, dried over Na₂SO₄, filtered and evaporated. Theresidue was purified by flash chromatography (hexane/EtOAc: 98:2→4:6).The residue was treated with hexane and Et₂O. The crystals obtained werefiltered off to give the title compound. t_(R): 1.26 min (LC-MS 1), ¹HNMR (400 MHz, <dmso>) 5 ppm 0.98 (s, 9H) 3.51-3.63 (m, 2H) 3.88 (dd,J=8.60, 4.30 Hz, 1H) 4.14 (dd, J=8.60, 4.69 Hz, 1H) 4.30-4.38 (m, 1H)7.37-7.50 (m, 6H) 7.57-7.65 (m, 4H) 7.71 (s, 1H).

Example 8(4S,5R)-342′-Amino-2-(D8-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the entire sequencedescribed for example 6, but using product from step 8.1 instead ofintermediate A and D-allo-Threoninol instead of D-threoninol. t_(R):0.79 min (LC-MS 1); ESI-MS: 464.5 [M+H]⁺ (LC-MS 1).

Step 8.1: 4(4,6-Dichloro-pyrimidin-2-yl)-D8-morpholine

The 2,4,6-trichloropyrimidine was dissolved in EtOH with Et₃N andD8-morpholine. The reaction mixture was stirred at RT for 1 H. It wasthen diluted with saturated NaHCO₃ solution and extracted twice withEtOAc. The organic extracts were combined and washed with brine. Then itwas dried over Na₂SO₄, filtered and concentrated. The residue waspurified by flash chromatography (hexane/EtOAc: 0% hexane→40%). t_(R):0.94 min (LC-MS 1); ESI-MS: 242.3/244.2 [M+H]⁺ (LC-MS 1).

Example 9(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-(2-hydroxy-ethyl)-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor example 6 but using product from step 9.1. The extraction wasperformed in DCM. The residue was purified by preparative HPLC (H₂O/ACN)then by flash chromatography (DCM/MeOH, 100:0→95:5). t_(R): 0.78 min(LC-MS 1); ESI-MS: 456.2 [M+H]⁺ (LC-MS 1).

Step 9.1:(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-(tert-butyl-diphenyl-silanyloxymethyl)-[1,3]oxazinan-2-one

The title compound was prepared in analogy to the procedure describedfor example 1 but using product from step 9.2. The reaction wasperformed at 120° C. for 15 min. The reaction mixture was dissolved inDCM and extracted with H₂O. The organic layer was dried (Na₂SO₄),filtered and concentrated. The residue was purified by flashchromatography (heptane/EtOAc, 8:2→4:6). t_(R): 1.54 min (LC-MS 1);ESI-MS: 694.3 [M+H]⁺ (LC-MS 1).

Step 9.2:(S)-4-(tert-Butyl-diphenyl-silanyloxymethyl)-3-(6-chloro-2-morpholin-4-yl-pyrimidin-4-yl)-[1,3]oxazinan-2-one

The title compound was prepared in analogy to the procedure describedfor step 2.2 but using product from step 9.3. The reaction was performedat 115° C. for 2.5 h. The mixture was concentrated. The residue waspurified by preparative HPLC (H₂O/ACN), then by flash chromatography(heptane/EtOAc, 9:1→0:100). t_(R): 1.49 min (LC-MS 1); ESI-MS: 567.3[M+H]⁺ (LC-MS 1).

Step 9.3:(S)-4-(tert-Butyl-diphenyl-silanyloxymethyl)-[1,3]oxazinan-2-one

To a solution of the product from step 9.4 (900 mg, 2.03 mmol) in THF(40 mL) under argon was added NaH 60% in oil (160 mg, 4.0 mmol) and themixture was stirred at rt for 4 h. The mixture was diluted with EtOAcand extracted with H₂O. The organic layer was dried (Na₂SO₄), filteredand concentrated. The residue was purified by flash chromatography(heptane/EtOAc, 100:0→0:100). t_(R): 1.23 min (LC-MS 1); ESI-MS: 370.2[M+H]⁺ (LC-MS 1).

Step 9.4:[(S)-1-(tert-Butyl-diphenyl-silanyloxymethyl)-3-hydroxy-propyl]-carbamicacid tert-butyl ester

To a solution of the product from step 9.5 (40 g, 73 mmol) in TBME (400mL) at 0° C. was added dropwise LiBH₄ (2M in THF, 74 mL, 146 mmol) andthe mixture was stirred at 0° C. for 10 min then warmed up at rt andstirred for 5 h. The reaction mixture was quenched with H₂O then with0.5M citric acid solution. The mixture was extracted with TBME. Theorganic layer was dried (MgSO₄), filtered and concentrated. The productwas used without further purification.

Step 9.5:(S)-3-tert-Butoxycarbonylamino-4-(tert-butyl-diphenyl-silanyloxy)-butyricacid benzyl ester

The title product was prepared in analogy to the procedure described forstep 6.4. R_(f): 0.7 (hexane/EtOAc, 8:2)

Step 9.6: (S)-3-tert-Butoxycarbonylamino-4-hydroxy-butyric acid benzylester

To a solution of Boc-L-aspartic acid-4-benzyl ester (100 g, 309 mmol) inDME (1.8 L) at −20° C. was added NMM (34 mL, 309 mmol) then dropwiseiso-butylchloroformate (40 mL, 309 mmol) and the mixture was stirred at−20° C. for 20 min. The reaction mixture was filtered and the filtratewas cooled to −20° C. NaBH₄ (17.5 g, 463 mmol) was added portionwise at−20° C. The mixture was allowed to warm and stirred at rt for 1 h. Themixture was quenched with a 20% citric acid solution and then extractedwith AcOEt. The organic layer was washed with NaHCO₃ solution, H₂O andbrine, dried (MgSO₄), filtered and concentrated. The product was usedwithout further purification for the next step.

Example 10(4S,5R)-3-[2′-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-hydroxymethyl-5-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the entire sequencedescribed for example 6, but using product from step 10.1 instead ofintermediate A and D-allo-Threoninol instead of D-threoninol. Themixture was dropwise added to a saturated solution of Na₂CO₃. After theaddition, a saturated solution of NaHCO₃ was added (the final pH wasaround 7-8. It was diluted with water and extracted with EtOAc. Theorganic phase was washed with brine, dried over Na₂SO₄, filtered andevaporated. The residue was purified by preparative HPLC (Waters SunFire C18, 30×100 mm, 5 um; 0.1% TFA-water/acetonitrile; gradientacetonitrile 5-100% in 20 min). The residue was recrystalised inEt2O/hexane (3/1). The crystals were filtered off and washed with hexaneto give the title compound. t_(R): 2.89 min (HPLC 1); ESI-MS: 470.3[M+H]⁺ (LC-MS 1); m.p. 217.7° C. (onset).

Step 10.1: (S)-4-(4,6-Dichloro-pyrimidin-2-yl)-3-methyl-morpholine

The 2,4,6-trichloropyrimidine (100 mg, 053 mmol) was dissolved indioxane (2 mL) with DIPEA (280 μL, 1.6 mmol) and (S)-3-methylmorpholine(54 mg, 0.53 mmol). The reaction mixture was heated at 130° C. undermicrowave irradiations for 15 min. It was then diluted with EtOAc andwashed with brine. The organic layer was dried over Na2SO4, filtered andconcentrated. The residue was purified by preparative HPLC (Waters SunFire C18, 30×100 mm, 5 um; 0.1% TFA-water/acetonitrile; gradientacetonitrile 5-100% in 20 min) to afford the title compound (45 mg,34%). t_(R): 3.70 min (HPLC 1); ESI-MS: 248.2/250.2 [M+H]⁺ (LC-MS 1).

Example 11 (For Comparison Purposes)3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-oxazolidin-2-one

The title compound was prepared in analogy to example 5 (including step5.1) but using the product from step 11.1. The reaction was performed at100° C. for 1 h. The extraction was performed in EtOAc. The residue waspurified by flash chromatography (heptane/EtOAc, 100:0→0:100). t_(R):0.87 min (LC-MS 1); ESI-MS: 412.4 [M+H]⁺ (LC-MS 1).

Step 11.1: 3-(6-Chloro-2-morpholin-4-yl-pyrimidin-4-yl)-oxazolidin-2-one

The title compound was prepared in analogy to the entire sequencedescribed for step 2.2 but using oxazolidin-2-one. t_(R): 0.93 min(LC-MS 1); ESI-MS: 285.5/287.4 [M+H]⁺ (LC-MS 1).

Example 12 Formic acid(4S,5R)-3-(2′-amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-methyl-2-oxo-oxazolidin-4-ylmethylester

The compound of example 18 (47 mg, 0.10 mmol) was dissolved in formicacid (80 μL, 2.09 mmol) and stored at 5° C. for 4 days. It was thenallowed to warm up to RT and it was stored for 2 days. It was then takenup with EtOAc and washed saturated NaHCO₃ solution and brine. Theorganic layer was dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by preparative HPLC (Waters Sun Fire C18, 30×100mm, 5 um; 0.1% TFA-water/acetonitrile; gradient acetonitrile 5-70% in 20min) to afford the title compound (57 mg, 80%). t_(R): 0.88 min (LC-MS1); ESI-MS: 484.4 [M+H]⁺ (LC-MS 1).

Example 13(S)-3-[2′-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the entire sequencedescribed for example 11, but using product from step 10.1 instead ofintermediate A and (S)-4-methyl-2-oxazolidinone instead ofoxazolidin-2-one. The residue was purified by flash chromatography(DCM/EtOH: 99.8/0.2→98/2) and then by preparative HPLC (Waters Sun FireC18, 30×100 mm, 5 um; 0.1% TFA-water/acetonitrile; gradient acetonitrile5-100% in 20 min) to afford the title compound (35 mg, 32%). t_(R): 0.98min (LC-MS 1); ESI-MS: 440.1 [M+H]⁺ (LC-MS 1).

Example 14(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor example 6 but using the product from step 14.1. The residue waspurified by flash chromatography (DCM/MeOH, 100:0→95:5). t_(R): 0.77 min(LC-MS 1); ESI-MS: 442.2 [M+H]⁺ (LC-MS 1).

Step 14.1:(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-(tert-butyl-diphenyl-silanyloxymethyl)-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor example 5 but using the product from step 14.2. The reaction wasperformed at 100° C. in an oil bath for 1 h. The extraction wasperformed in EtOAc. The residue was purified by flash chromatography(heptane/EtOAc, 100:0→0:100). t_(R): 1.40 min (LC-MS 1); ESI-MS: 680.3[M+H]⁺ (LC-MS 1).

Step 14.2:(S)-5-(tert-Butyl-diphenyl-silanyloxymethyl)-3-(6-chloro-2-morpholin-4-yl-pyrimidin-4-yl)-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 2.2 but using the product from step 14.3. After extraction, theresidue was purified by flash chromatography (heptane/EtOAc,100:0→40:60). t_(R): 1.49 min (LC-MS 1); ESI-MS: 553.3 [M+H]⁺ (LC-MS 1).

Step 14.3: (S)-5-(tert-Butyl-diphenyl-silanyloxymethyl)-oxazolidin-2-one

To a solution of the product from step 14.4 (2.72 g, 8.27 mmol) and Et₃N(2.88 mL, 20.67 mmol) in DCM under argon was added dropwise triphosgene(982 mg, 3.31 mmol) and the mixture was stirred at rt for 5 h. Thereaction mixture was quenched with a NH₄Cl solution. The organic layerwas dried (Na₂SO₄), filtered and concentrated. The residue was purifiedby flash chromatography (heptane/EtOAc, 100:0→0:100). t_(R): 1.21 min(LC-MS 1); ESI-MS: 373.2 [M+H]⁺ (LC-MS 1).

Step 14.4: (S)-1-Amino-3-(tert-butyl-diphenyl-silanyloxy)-propan-2-ol

The title compound was prepared in analogy to the procedure describedfor step 6.4 but using (S)-3-aminopropane-1,2-diol and using Et₃Ninstead of imidazole. After extraction, the residue was purified byflash chromatography (DCM/MeOH, 100:0→90/10). t_(R): 0.93 min (LC-MS 1);ESI-MS: 330.2 [M+H]⁺ (LC-MS 1).

Example 15(4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-4-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the entire sequencedescribed for example 6, but using product from step 15.1 instead ofD-threoninol. t_(R): 0.98 min (LC-MS 1); ESI-MS: 344.3 [M+H]⁺ (LC-MS 1).

Step 15.1: (2R,3S)-3-Amino-butane-1,2-diol

The title compound was prepared in analogy to the procedure of step19.1, but using product from step 15.2. The residue was purified byflash chromatography (DCM/EtOH: 99.8/0.2→98/2) and then by preparativeHPLC (Waters Sun Fire C18, 30×100 mm, 5 um; 0.1% TFA-water/acetonitrile;gradient acetonitrile 5-100% in 20 min) to afford the title compound (35mg, 32%). t_(R): 0.98 min (LC-MS 1); ESI-MS: 440.1 [M+H]⁺ (LC-MS 1).

Step 15.2:N-Benzyl-N—[(S)-1-((R)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-hydroxylamine

The title compound is the second isomer formed during the step 19.2(1.07 g, 57%). t_(R): 0.91 min (LC-MS 1); ESI-MS: 252.2 [M+H]⁺ (LC-MS1).

Example 16(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-methyl-oxazolidin-2-one

The title compound was prepared in analogy to example 11, but usingproduct from step 16.1 instead of (R)-4-methyl-2-oxazolidinone. Thereaction was performed at 100° C. for 1 H. The reaction mixture wastaken up with EtOAc. It was washed twice with saturated NaHCO₃ solutionand once with brine. The organic layer dried over sodium sulfate. Theresidue was purified by flash chromatography (heptane/EtOAc: 0%→85%EtOAc) to give the title compound (9.6 mg, 58%). t_(R): 0.94 min (LC-MS1); ESI-MS: 426.2 [M+H]⁺ (LC-MS 1).

Step 16.1: (S)-5-Methyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 6.3, but using (S)-1-aminopropan-2-ol. The reaction wasperformed at RT for 3 H. The reaction was quenched with saturated NH4Clsolution (10 mL) and stirred 10 min at RT. The water layer was separatedand the organic layer washed with water. The combined aqueous layer wereextracted 3× with DCM. The combined organic layer were dried overNa₂SO₄, filtered and concentrated. The residue was purified by flashchromatography (heptane/EtOAc: 0%→100% EtOAc) to give the title compound(38 mg, 9%). ¹H NMR (400 MHz, <DMSO>) δ ppm 1.27 (d, J=6.25 Hz, 3H)2.94-3.08 (m, 1H) 3.48-3.58 (m, 1H) 4.62 (m, 1H) 7.37 (br. s., 1H)

Example 17(S)-3-(2′-amino-2-D8-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-4-methyloxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor example 4 but using the product from step 17.1. After completion,the reaction mixture was filtered through celite and concentrated. Theresidue was purified by flash chromatography (DCM/EtOH, 99.5:0.5→98:2).The residue was triturated in DCM and washed with hexane to afford thetitle compound. t_(R): 0.89 min (LC-MS 1); ESI-MS: 434.4 [M+H]⁺ (LC-MS1); R_(f): 0.67 (DCM/EtOH, 95:5).

Step 17.1:(S)-3-(6-Chloro-2-D8-morpholin-4-yl-pyrimidin-4-yl)-4-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 2.2 but using the product from step 8.1 and(S)-4-methyl-2-oxazolidinone. The extraction was performed in DCM. Theresidue was purified by flash chromatography (heptane/EtOAc, 9:1→7:3).t_(R): 0.97 min (LC-MS 1); ESI-MS: 307.3/309.3 [M+H]⁺ (LC-MS 1).

Example 18(4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the entire sequencedescribed for example 6, but using D-allo-Threoninol instead ofD-threoninol. The reaction was performed at RT for 33H. The mixture wasdropwised added to a saturated solution of Na₂CO₃. After the addition,the pH was around 7-8. It was diluted with water and extracted withEtOAc. The organic phase was washed with brine, dried over Na₂SO₄,filtered and evaporated. The residue was purified by preparative HPLC(Waters Sun Fire C18, 30×100 mm, 5 um; 0.1% TFA-water/acetonitrile;gradient acetonitrile 5-60% in 20 min). The fractions were combined andbasified with 5% NaHCO₃ solution. The product precipitated and wasfiltered off. To eliminate residual Palladium the product was dissolvedin DCM/MeOH (4/1) and was passed through an SPE cartridge of MP-Thiolfrom polymerlabs and then the solvent was evaporated. A number ofbatches were produced based on this method, and several worked-up toprovide crystalline material as follows.

Batch A:

For the preparation of this batch the product was not passed through anSPE cartridge of MP-Thiol from polymerlabs. The pure fractions obtainedafter the preparative HPLC were combined and treated with NaHCO3. TheCH₃CN was evaporated whereupon the product crystallized. The product wascollected by filtration washed with water and dried to give the titlecompound as a white solid. m.p. 221.3° C. (onset).

Batch B:

For the preparation of this batch the product was not passed through anSPE cartridge of MP-Thiol from polymerlabs. The pure fractions obtainedafter the preparative HPLC were combined and treated with solid NaHCO3.The CH₃CN was evaporated whereupon the product crystallized. The aqueousmixture was kept for 1 h in the refrigerator, filtered, washed withwater and dried under HV overnight to afford a white solid, m=298 mg.m.p. 249.9° C. (onset)

Batch C:

For the preparation of this batch the product was not passed through anSPE cartridge of MP-Thiol from polymerlabs. The pure fractions obtainedafter the preparative HPLC were combined and evaporated. The residue wastaken up in CH₃CN and then water containing 0.1% TFA was added followedby solid NaHCO₃. The solution is concentrated and the precipitate isfiltered, washed with water and dried to give the title product as awhite solid. m.p. 237.9° C. (onset)

Batch D:

After the passage through the SPE cartridge of MP-Thiol from polymerlabsthe solvent was evaporated. The residue was dissolved in CH₃CN and thendiluted with the same amount of water. The CH₃CN was evaporated and justbefore crystallization of the product, some crystals of Batch B wereadded. CH₃CN was then completely evaporated and the suspension wascooled in the fridge. It was then filtered, collected and dried under HVovernight to afford a the title product as a white solid, m.p. 259.0° C.(onset)

Batch E:

Same procedure as described for Batch C but some crystals of Batch Dwere added to induce crystallisation. The title product was obtained asa white solid, m.p. 258.8° C. (onset).

Example 19(4S,5S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-4-methyl-oxazolidin-2-one

The title compound was prepared in analogy to the entire sequencedescribed for example 6, but using product from step 19.1 instead ofD-threoninol. The reaction was performed at RT for 16H 30. The mixturewas dropwised added to a saturated solution of Na₂CO₃. After theaddition, it was diluted with water and extracted twice with EtOAc. Theorganic phase was washed with brine, dried over Na₂SO₄, filtered andevaporated. The residue was purified by preparative HPLC (Waters SunFire C18, 30×100 mm, 5 um; 0.1% TFA-water/acetonitrile; gradientacetonitrile 5-80% in 20 min) to give the title compound (16.3 mg, 75%).t_(R): 2.79 min (HPLC 1); ESI-MS: 456.1 [M+H]⁺.

Step 19.1: (2S,3S)-3-Amino-butane-1,2-diol

The product from step 19.2 was stirred in HCl in solution in EtOH for 2H at RT. The solvent was removed to give the title compound as an HClsalt (303 mg, 100%). ¹H NMR (400 MHz, <dmso>) δ ppm 1.08 (d, J=6.65 Hz,3H) 3.18-3.33 (m, 1H) 3.33-3.45 (m, 1H) 3.61-3.72 (m, 1H) 7.85 (br. s.,2H)

Step 19.2: (S)-1-((S)-2,2-Dimethyl-[1,3]dioxolan-4-yl)-ethylamine

The product from step 19.3 (product 2, last eluting, 538 mg, 2.14 mmol)was dissolved in AcOH (25 mL) with Pd/C (100 mg) and the reactionmixture was stirred at RT for 11H under H₂ conditions. It was thenfiltered over Celite and then the solvent was removed to give the titlecompound (311 mg, 100%). NMR (400 MHz, <dmso>) δ ppm 0.90-1.04 (m, 3H)1.16-1.38 (m, 6H) 2.76-2.90 (m, 1H) 3.75 (dd, J=13.86, 7.22 Hz, 2H) 3.90(br. s., 1H); t_(R): 3.13 min (HPLC 1).

Step 19.3:N-Benzyl-N—[(R)-1-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-hydroxylamineandN-Benzyl-N—[(S)-1-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-ethyl]-hydroxylamine

To a well stirred solution of 1.48 g (6.29 mmol) of the product fromstep 19.4 in 80 ml Et₂O was added 6.29 ml (6.29 mmol) 1M Et₂AlCl inhexanes in one portion and stirring was continued for 15 min. Themixture was then cooled to −60° C. and treated with 6.29 ml (18.87 mmol)3M Methylmagnesium bromide in Et₂O. The mixture was stirred for 2 h at−60° C. and then allowed to slowly warm up to RT under stirring duringthe night. After that, the reaction was treated with NaOH (2M, 40 ml).After stirring for 15 min at RT, the mixture was extracted 3×120 withEt₂O. The combined organic layers were dried over Na₂SO₄, filtered andconcentrated under vacuum. The residue is dissolved in MeOH and purifiedby reverse phase prep. HPLC in 8 injections (H₂O[+0.1% TFA]/CH₃CN 97:3to 50:50 in 20 min.):

-   -   Fractions 1-3 were collected together and basified with ˜2 g        NaHCO₃, before being concentrated. The resulting layer was        extracted with 2×150 ml Et₂O and the combined organic layers        were dried over Na2SO4, filtered and evaporated to dryness to        give 1.01 g of a colorless oil, which slowly crystallizes (˜99%        pure by 1HNMR; HPLC Rt=2.36; ESI-MS: 252.2 [M+H]⁺ (LC-MS        1))→Product 1    -   Fractions 5-7 were collected together and basified with ˜2 g        NaHCO₃, before being concentrated. The resulting suspension was        extracted with 2×150 ml Et₂O and the combined organic layers are        dried over Na₂SO₄, filtered and evaporated to dryness to give        363 mg of a white solid (˜99% pure by 1 HNMR; HPLC Rt=2.44;        ESI-MS: 252.3 [M+H]+(LC-MS 1))→Product 2.

Step 19.4:(S,Z)-N-((2,2-dimethyl-1,3-dioxolan-4-yl)methylene)-1-phenylmethanamineoxide

1.50 g (11.53 mmol) (R)-2,2-Dimethyl-1.3-dioxolane-4-carboxaldehyde(Fluorochem, Hadfield, UK) was dissolved in 60 ml of DCM and treatedwith 1.64 g (11.53 mmol) sodium sulfate. The reaction mixture wasflushed with argon and treated with a solution of 1.42 g (11.53 mmol)N-benzyl-hydroxylamine (prepared from the commercially availablehydrochloride salt) in 20 ml of CH2Cl2. The reaction mixture was stirredunder argon at RT for 16.5 h and then filtered. Silicagel was added tothe filtrate is and preabsorbed, before being purified by chromatographyon silicagel (gradient: Heptane/EtOAc 0%-100% in 30 min). Fr. 20-80 werecollected and evaporated to dryness and dried under vacuum overnight togive 1.48 g of a white solid (˜100% pure by HPLC, Rt=1.43); ESI-MS:236.2 [M+H]+(LC-MS 1))

Example 20(R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-oxazolidin-2-one

The title compound was prepared in analogy to the entire sequencedescribed for example 6, but using (R)-3-aminopropane-1,2-diol insteadof D-threoninol. The reaction was performed at RT for 16H. Then thereaction mixture was quenched carefully with NaHCO₃. Then it was dilutedwith EtOAc and washed twice with saturated NaHCO3 solution and once withbrine. The organic layer was dried over Na₂SO₄, filtered and evaporated.The residue was purified by flash chromatography (DCM/EtOH: 0%→10% MeOH)to give the title compound (22.8 mg, 38%). t_(R): 0.77 min (HPLC 1);ESI-MS: 442.2 [M+H]⁺ (LC-MS 1).

Example 21(3aR,6aR)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-tetrahydrofuro[3,4-d]oxazol-2(3H)-one

A solution(3aR,6aR)-3-(6-chloro-2-morpholinopyrimidin-4-yl)tetrahydrofuro[3,4-d]oxazol-2(3H)-one(100 mg, 0.306 mmol), intermediate B (115 mg, 0.398 mmol), K₃PO₄ (195mg, 0.918 mmol) and PdCl₂(dppf)-CH₂Cl₂ (25 mg, 0.031 mmol) in DME/H₂O(2.2 mL) under argon was stirred at 80° C. for 1.5 h. The mixture wasdiluted in EtOAc and extracted with saturated NaHCO₃. The organic layerwas washed with H₂O and brine, dried over MgSO₄, filtered andconcentrated. The residue was purified by flash chromatography(hexane-EtOAc 70:30→0:100) to afford the title compound as a colorlesssolid (88 mg, 62%): t_(R)=0.84 min (LC-MS 3); ESI-MS: 454 [M+H]⁺ (LC-MS3).

Step 21.1:(3aR,6aR)-3-(6-Chloro-2-morpholinopyrimidin-4-yl)tetrahydrofuro[3,4-d]oxazol-2(3H)-one

To a solution of (3aR,6aR)-tetrahydrofuro[3,4-d]oxazol-2(3H)-one (500mg, 3.87 mmol), 4-(4,6-dichloropyrimidin-2-yl)morpholine (1088 mg, 4.65mmol) and Cs₂CO₃ (2.14 g, 6.58 mmol) in dioxane (20 mL) was added afterdegassing with argon 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene(157 mg, 0.271 mmol) and Pd₂(dba)₃ (70.9 mg, 0.077 mmol) and thereaction mixture was heated for 6 h at 85° C. The reaction mixture wasadded to 10% aqueous NaHCO₃ solution and extracted with EtOAc. Combinedextracts were washed with brine, dried over MgSO4, filtered andconcentrated. The crude product was triturated in MeOH overnight,filtered off and dried to afford the title compound as a colorless solid(1.12 g, 87%): t_(R)=0.92 min (LC-MS 3); ESI-MS: 327, 329 [M+H]⁺ (LC-MS3).

Step 21.2: (3aR,6aR)-Tetrahydrofuro[3,4-d]oxazol-2(3H)-one

To a solution of (3R,4R)-4-aminotetrahydrofuran-3-ol (1.1 g, 7.88 mmol)and DIEA (4.54 ml, 26.0 mmol) in CH₂Cl₂ (30 mL) was added(bis(trichloromethyl) carbonate (1.75 g, 5.91 mmol) dissolved in CH₂Cl₂(5 mL) at RT over a period of 30 min. After stirring for 0.5 h at 25°C., the reaction mixture was added to aqueous K₂CO₃ solution, stirredfor 1 h and the CH₂Cl₂ was evaporated. The aqueous phase was washed withEt₂O and afterwards evaporated to dryness. The residue was trituratedwith EtOH/THF 1:1, the inorganic salts removed by filtration through aplug of silica gel, and the filtrate was concentrated to provide thetitle compound as a beige solid (930 mg, 90%): ESI-MS: 147 [M+NH]⁺.

Example 22(3aR*,6R*,6aR*)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-hydroxyhexahydro-2H-cyclopenta[d]oxazol-2-one

To a solution of(3aR*,6R*,6aR*)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-((tert-butyldimethylsilyl)oxy)hexahydro-2H-cyclopenta[d]oxazol-2-one(810 mg, 1.253 mmol) in THF (12 mL), was added dropwise 1M TBAF in THF(1.0 mL, 1.0 mmol) at 0° C. The reaction mixture was stirred for 30 minat 0° C. and 2 h at RT before evaporation. The residue was purified byflash chromatography (hexane-THF 60:40→0:100). The residue wastriturated in Et₂O, filtered and dried. The residue was purified bypreparative HPLC (Waters Sun Fire C18, 30×100 mm, 5 um; 0.1%TFA-water/acetonitrile; gradient acetonitrile 5-100% in 20 min) toafford the title compound (430 mg, 72%); t_(R)=0.93 min (UPLC 1),t_(R)=0.81 min (LC-MS 3); ESI-MS: 468 [M+H]⁺ (LC-MS 3).

Step 22.1:(3aR*,6R*,6aR*)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-((tert-butyldimethylsilyl)oxy)hexahydro-2H-cyclopenta[d]oxazol-2-one

The title compound was prepared in analogy to the procedure describedfor example 21 from(3aR*,6R*,6aR*)-6-((tert-butyldimethylsilyl)oxy)-3-(6-chloro-2-morpholinopyrimidin-4-yl)hexa-hydro-2H-cyclopenta[d]oxazol-2-oneand intermediate B and using Pd(PPh₃)₄ instead of PdCl₂(dppf)-CH₂Cl₂ andNa₂CO₃ instead of K₃PO₄ to afford the title compound aftercrystallization from EtOAc/hexane as white solid: t_(R)=1.62 min (UPLC1), t_(R)=1.47 min (LC-MS 3); ESI-MS: 582 [M+H]⁺ (LC-MS 3).

Step 22.2:(3aR*,6R*,6aR*)-6-((tert-butyldimethylsilyl)oxy)-3-(6-chloro-2-morpholinopyrimidin-4-yl)hexahydro-2H-cyclopenta[d]oxazol-2-one

The title compound was prepared in analogy to the procedure describedfor step 21.1 from(3aR*,6R*,6aR*)-6-((tert-butyldimethylsilyl)oxy)hexahydro-2H-cyclopenta[d]oxazol-2-oneand intermediate A: t_(R)=1.76 min (UPLC 1), t_(R), 1.58 min (LC-MS 3);ESI-MS: 455, 457 [M+H]⁺ (LC-MS 3).

Step 22.3:(3aR*,6R*,6aR*)-6-((tert-Butyldimethylsilyl)oxy)hexahydro-2H-cyclopenta[d]oxazol-2-one

To a suspension of(3aR*,6R*,6aR*)-6-((tert-butyldimethylsilyl)oxy)-3-((2-nitrophenyl)sulfonyl)-hexahydro-2H-cyclopenta[d]oxazol-2-one(1.47 g, 3.32 mmol) and Cs₂CO₃ (2.164 g, 6.64 mmol) in DMF (25 mL) wasadded N-acetyl-L-cysteine (0.921 g, 5.65 mmol) and the reaction mixturewas stirred for 16 h at RT. The reaction mixture was evaporated and theresidue suspended in saturated NaHCO3 solution and extracted with EtOAc.Combined extracts were washed with brine, dried over MgSO4, filtered andconcentrated. The title compound was obtained after purification byflash chromatography (heptane/EtOAc 90:10→50:50) as a yellow oil (0.84g, 98%): TLC (heptane/EtOAc 1:1) R_(f)=0.28.

Step 22.4:(3aR*,6R*,6aR*)-6-((tert-Butyldimethylsilyl)oxy)-3-((2-nitrophenyl)sulfonyl)hexahydro-2H-cyclopenta[d]oxazol-2-one

To a solution of(3aR*,6R*,6aR*)-6-hydroxy-3-((2-nitrophenyl)sulfonyl)hexahydro-2H-cyclo-penta[d]oxazol-2-one(1.2 g, 3.66 mmol) and 2,6-lutidine (0.851 mL, 7.31 mmol) in CH₂Cl₂ (25mL) was added dropwise at 0° C. the tert-butyldimethylsilyltrifluoromethanesulfonate (1.091 mL, 4.75 mmol). The reaction mixturewas stirred for 1 h at 0° C. followed by 2 h at RT. The reaction mixturewas diluted with CH₂Cl₂ and washed with 20% aqueous NaH₂PO₄ solution andH₂O, dried over MgSO₄, filtered and concentrated. The title compound wasobtained after crystallization from EtOAc/heptane as white solid (1.5 g,88%): TLC (heptane/EtOAc 1:1) R_(f)=0.54; t_(R)=1.58 min (UPLC 1),t_(R)=1.43 min (LC-MS 3); ESI-MS: 460 [M+NH₄]⁺ (LC-MS 3).

Step 22.5:(3aR*,6R*,6aR*)-6-Hydroxy-3-((2-nitrophenyl)sulfonyl)hexahydro-2H-cyclopenta[d]-oxazol-2-one

To a solution of tert-butyl(1R*,2S*,5S*)-6-oxabicyclo[3.1.0]hexan-2-yl((2-nitrophenyl)sulfonyl)-carbamate(2.0 g, 5.10 mmol) in MeOH (40 mL) was added Amberlyst 15 (4.0 g) andthe resulting suspension was stirred for 1.5 h at 25° C. The reactionmixture was filtered and concentrated. The title compound was obtainedafter purification by flash chromatography (heptane-EtOAc 90:10→EtOAc)as a beige solid (1.24 g, 73%): TLC (heptane/EtOAc 1:2) R_(f)=0.36;t_(R)=0.80 min (UPLC 1), t_(R), 0.77 min (LC-MS 3); ESI-MS: 346 [M+NH₄]⁺(LC-MS 3).

Step 22.6: (tert-Butyl(1R*,2S*,5S*)-6-oxabicyclo[3.1.0]hexan-2-yl((2-nitrophenyl)sulfonyl)-carbamate

To a suspension of tert-butylcyclopent-2-en-1-yl((2-nitrophenyl)sulfonyl)carbamate (2.75 g, 7.46mmol) and NaHCO₃ (1.254 g, 14.93 mmol) in CH₂Cl₂ (60 mL) was added inone portion meta-chloroperoxybenzoic acid (2.58 g, 14.93 mmol). Theresulting reaction mixture was stirred overnight at RT. The reactionmixture was diluted with CH₂Cl₂ and washed with 20% aqueous Na₂SO₃solution, saturated NaHCO₃ solution and water. The organic phase wasdried over MgSO₄ and concenrated. The title compound was obtained aftercrystallization from EtOAc as white crystals (2.01 g, 68%): TLC(heptane-EtOAc 1:1) R_(f)=0.48; t_(R)=1.20 min (UPLC 1), t_(R)=1.12 min(LC-MS 3); ESI-MS: 329 [M-isobutylene]⁺ (LC-MS 3).

Step 22.7: (tert-butylcyclopent-2-en-1-yl((2-nitrophenyl)sulfonyl)carbamate

To a suspension of triphenylphosphine (3.09 g, 11.77 mmol), tert-butyl2-nitrophenylsulfonyl-carbamate (3.40 g, 11.23 mmol) andcyclopent-2-enol (0.900 g, 10.70 mmol) in toluene (60 mL) was addeddropwise at −20° C. the diethylazodicarboxylate (1.948 mL, 12.30 mmol).The reaction mixture was stirred at −20° C. for 2 h followed by 3 h at0° C. The reaction mixture was concentrated and the title compound wasobtained after purification by flash chromatography (heptane/EtOAc95:5→3:1) as a white solid (2.79 g, 67%): t_(R)=1.34 min (UPLC 1),t_(R), 1.24 min (LC-MS 3); ESI-MS: 386 M+NH₄]⁺ (LC-MS 3).

Example 22A First Eluting Enantiomer on Chiralpak AD of (3aR,6R,6aR)-and(3aS,6S,6aS)-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-hydroxyhexahydro-2H-cyclopenta[d]oxazol-2-one

Absolute stereochemistry not determined.

The title compound was obtained after preparative chiral HPLC separationof the racemic product of example 22. (Column: Chiralpak AD 20 μm 5×500mm. Flow 70 mL/min. heptane/EtOH/DEA 20:80:0.01). t_(R): 17.7 min(Column: Chiralpak AD-H, 4.6×250 mm. Flow 1.2 mL/min. heptane/EtOH70:30).

Example 22B Second Eluting Enantiomer on Chiralpak AD of (3aR,6R,6aR)-and(3aS,6S,6aS)-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-hydroxyhexahydro-2H-cyclopenta[d]oxazol-2-one

Absolute stereochemistry not determined.

The title compound was obtained after preparative chiral HPLC separationof the racemic product of example 22. (Column: Chiralpak AD 20 μm 5×500mm. Flow 70 mL/min. heptane/EtOH/DEA 20:80:0.01). t_(R): 23.3 min(Column: Chiralpak AD-H, 4.6×250 mm. Flow 1.2 mL/min. heptane/EtOH70:30).

Example 23(4S,5R)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-hydroxyethyl)-4-methyloxazolidin-2-one

To a solution of(4S,5R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-4-methyloxazolidin-2-one(2.1 g, 3 mmol) in THF (20 mL), was added dropwise 1M TBAF in THF (3 mL,3 mmol) at 0° C. The reaction mixture was stirred for 1 h at 0° C.before evaporation. The residue was purified by flash chromatography(hexane/EtOAc/MeOH 90:10:1→0:100:10). The purified product wasre-crystallized from MeOH to afford the title compound as a white solid(1.17 g, 83%): t_(R)=0.80 min (UPLC 1), t_(R)=0.82 min (LC-MS 3);ESI-MS: 470 [M+H]⁺ (LC-MS 3).

Step 23.1:(4S,5R)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-4-methyloxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor example 22.1 from4S,5R)-5-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-3-(6-chloro-2-morpholinopyrimidin-4-yl)-4-methyloxazolidin-2-oneand intermediate B to afford the title compound after crystallizationfrom MeOH: t_(R)=1.72 min (UPLC 1), t_(R)=1.55 min (LC-MS 3); ESI-MS:708 [M+H]⁺ (LC-MS 3).

Step 23.2:(4S,5R)-5-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-3-(6-chloro-2-morpholinopyrimidin-4-yl)-4-methyloxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 21.1 from a 4:1 mixture of the (4S,5R)- and(4S,5S)-diastereoisomer of5-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-3-(6-chloro-2-morpholinopyrimidin-4-yl)-4-methyloxazolidin-2-oneand intermediate A to afford after removal of the(4S,5S)-diastereoisomer by two re-crystallizations from THF/MeOH onlythe (4S,5R)-diastereoisomer of the title compound: t_(R)=1.88 min (UPLC1), t_(R)=1.64 min (LC-MS 3); ESI-MS: 581, 583 [M+H]⁺ (LC-MS 3); ¹H NMR(400 MHz, DMSO-d₆): δ 1.08 (s, 9H), 1.36 (d, 3H), 2.02 (m, 2H),3.70-3.90 (m, 10H), 4.82 (m, 2H), 7.40-7.50 (m, 6H), 7.51 (s, 1H), 7.67(m, 4H).

Step 23.3: (4S,5R)- and(4S,5S)-5-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-4-methyloxazolidin-2-one

To a solution of a 4:1 mixture of the (4S,5R)- and(4S,5S)-diastereoisomer of5-(2-((tert-butyl-diphenylsilyl)oxy)ethyl)-3-(4-methoxybenzyl)-4-methyloxazolidin-2-one(4.1 g, 7.8 mmol) in acetonitrile (40 mL) was added a solution of(NH₄)₂Ce(NO₃)₆ (10.71 g, 19.5 mmol) in H₂O (20 mL) at 0° C. The reactionmixture was stirred for 4 h at 0-5° C. The mixture was added toice-water and the product extracted with EtOAc. Combined extracts werewashed with saturated NaHCO₃ solution and brine, dried over MgSO₄,filtered and concentrated. The title compound was obtained afterpurification by flash chromatography (heptane/EtOAc 10:1→EtOAc) as a 4:1mixture of diastereomers (2.2 g, 71%): TLC (hexane-EtOAc 1:1)R_(f)=0.23; t_(R)=1.47 min (UPLC 1), t_(R), 1.33 min (LC-MS 3); ESI-MS:401 [M+NH4]⁺ (LC-MS 3).

Step 23.4: (4S,5R)- and(4S,5S)-5-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-3-(4-methoxybenzyl)-4-methyloxazolidin-2-one

To a solution of a 4:1 mixture of the (4S,5R)- and (4S,5S)-diastereomersof 5-(2-hydroxyethyl)-3-(4-methoxybenzyl)-4-methyloxazolidin-2-one (2.65g, 10 mmol) in DMF (30 mL) was added imidazole (1.73 g, 25 mmol) andTBDPSCl (3.57 g, 13 mmol) at 0-5° C. The reaction mixture was allowed towarm to RT and was stirred overnight at RT. The reaction mixture wasconcentrated and the residual oil was dissolved in TBME and washed with10% aqueous KHSO₄ solution, H₂O, saturated NaHCO₃ solution and brine,dried over MgSO₄, filtered and concentrated. The title compound wasobtained after purification by flash chromatography (heptane/EtOAc20:1→2:1) as a 4:1 mixture of diastereomers (4.18 g, 80%): TLC(hexane/EtOAc 3:1) R_(f)=0.27; t_(R)=1.70 min (UPLC 1), t_(R), 1.52 min(LC-MS 3); ESI-MS: 526 [M+Na]⁺ (LC-MS 3).

Step 23.5: (4S,5R)- and(4S,5S)-5-(2-hydroxyethyl)-3-(4-methoxybenzyl)-4-methyloxazolidin-2-one

A 4:1 mixture of (4S,5R)- and(4S,5S)-5-allyl-3-(4-methoxybenzyl)-4-methyloxazolidin-2-one (2.67 g, 10mmol) in CH₂Cl₂-MeOH 2:1 (40 mL) was ozonated at −78° C. After completeozonide formation NaBH₄ (0.57 g, 15 mmol) was added and the reactionmixture was allowed to warm to RT and stirred for 2 h at RT. Thereaction mixture was added to a 20% aqueous K₂CO₃ solution and theproduct was extracted with EtOAc. Combined extracts were washed withbrine, dried over MgSO₄, filtered and concentrated to afford the titlecompound as a light yellow oil (2.65 g, 99%): TLC (EtOAc) R_(f)=0.28;t_(R)=0.68 min (UPLC 1), t_(R), 0.69 min (LC-MS 3); ESI-MS: 266 [M+H]⁺(LC-MS 3).

Step 23.6: (4S,5R)- and(4S,5S)-5-allyl-3-(4-methoxybenzyl)-4-methyloxazolidin-2-one

To a solution of a 4:1 mixture of benzyl((2S,3R)-3-hydroxyhex-5-en-2-yl)(4-methoxybenzyl)-carbamate and benzyl((2S,3S)-3-hydroxyhex-5-en-2-yl)(4-methoxybenzyl)carbamate (3.55 g, 9.5mmol) in THF (60 mL) was added under argon at −50° C. a 1M solution ofNaHMDS in THF (10.5 mL). After stirring the reaction mixture for 0.5 hat −40° C. the mixture was added to cold 10% aqueous KHSO₄ solution andthe product was extracted with EtOAc. Combined extracts were washed withbrine, dried over MgSO₄, filtered and concentrated. The title compoundwas obtained after purification by flash chromatography (hexane/EtOAc10:1→1:1) as a colorless oil (2.4 g, 97%): TLC (hexane/EtOAc 1:1)R_(f)=0.42; t_(R)=1.03 min (UPLC 1), t_(R), 0.99 min (LC-MS 3); ESI-MS:262 [M+H]⁺ (LC-MS 3); ¹H NMR (400 MHz, DMSO-d₆): δ 1.06 (d, 2.4H), 1.13(d, 0.6H), 2.30-2.40 (m, 2H), 3.25 (m, 0.2H), 3.66 (m, 0.8H, strong NOEto signal at 4.54), 3.75 (s, 3H), 4.07 (d, 1H), 4.10 (m, 0.2H), 4.48 (d,1H), 4.54 (m, 0.8H), 5.05-5.20 (m, 2H), 5.23 (m, 0.2H), 5.78 (m, 0.8H),6.92 (d, 2H), 7.22 (d, 0.4H), 7.24 (d, 1.6H).

Step 23.7: (2S,3R)- and (25,35)-benzyl4-methoxybenzyl(1-oxopropan-2-yl)carbamate

To a solution of (S)-benzyl 4-methoxybenzyl(1-oxopropan-2-yl)carbamate(3.86 g, 10 mmol) in THF (30 mL) was added zinc dust (1.64 g, 25 mmol),saturated aqueous NH₄Cl solution (5 mL) and allyl bromide (2.2 mL, 25mmol) and the reaction mixture was stirred for 1 h at 25-30° C. Thereaction mixture was diluted with H₂O and the product was extractedEtOAc. Combined extracts were washed with 10% aqueous KHSO₄ solution,H₂O, NaHCO₃ and brine, dried over MgSO4, filtered and concentrated. Thetitle compound was obtained after purification by flash chromatography(hexane/EtOAc 20:1→2:1) as a colorless oil (3.5 g, 97%): t_(R)=1.25 minand 1.27 min (UPLC 1) (4:1 mixture of (2S,3R)- and(25,35)-diastereoisomer), TLC (hexane/EtOAc 1:1) R_(f)=0.51; t_(R), 1.68min and 1.69 min (LC-MS 3); ESI-MS: 370 [M+H]⁺ (LC-MS 3).

Step 23.8: (S)-benzyl 4-methoxybenzyl(1-oxopropan-2-yl)carbamate

To a solution of (S)-benzyl(1-hydroxypropan-2-yl)(4-methoxybenzyl)carbamate (11.8 g, 35.5 mmol) inCH₂Cl₂ was added NaHCO₃ (3.28 g, 39 mmol), KBr (0.25 g, 2.1 mmol) andTEMPO 0.168 g, 1.07 mmol). The reaction mixture was cooled to 0-5° C.and the 5% aqueous NaClO solution (85 mL, 71 mmol) was added within 30min. After stirring for 1 h at 0-5° C. the reaction mixture was added toNa₂S₂O₃ solution and the product was extracted with EtOAc. Combinedextracts were washed with aqueous NaH₂PO₄ solution, H₂O and brine, driedover MgSO₄, filtered and concentrated to provide the title compound as ayellow oil (11.2 g, 96%): TLC (hexane/EtOAc 1:1) R_(f)=0.55; t_(R)=1.29min (UPLC 1), t_(R), 1.15 min (LC-MS 3); ESI-MS: 328 [M+H]⁺ (LC-MS 3).

Step 23.10: (S)-benzyl (1-hydroxypropan-2-yl)(4-methoxybenzyl)carbamate

To a solution of(S)-2-(((benzyloxy)carbonyl)(4-methoxybenzyl)amino)propanoic acid[439589-23-8] (16 g, 41.9 mmol) in THF (150 mL) was slowly added underargon borane dimethylsulfide (8.4 mL, 84 mmol) at 0-5° C. The reactionmixture was stirred for 1 h at 0-5° C. followed by 3 h at 40-45° C.Excess borane was destroyed by careful addition of MeOH and the reactionmixture was evaporated 3× with 200 mL MeOH and 2× with CHCl₃. The titlecompound was obtained after drying as a colorless oil (13.7 g, 99%): TLC(hexane/EtOAc 1:1) R_(f)=0.22; t_(R)=1.06 min (UPLC 1), t_(R), 1.02 min(LC-MS 3); ESI-MS: 330 [M+H]⁺ (LC-MS 3).

Example 24 First eluting diastereoisomer on the LC-MS 3 of(4S,5R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-((R)-2-hydroxypropyl)-4-methyloxazolidin-2-one

Absolute stereochemistry of 2-hydroxypropyl moiety not determined,(R)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor example 23 from(4S,5R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-((R)-2-((tert-butyl-diphenylsilyl)oxy)propyl)-4-methyloxazolidin-2-oneand TBAF to afford after purification by flash chromatography(hexane/EtOAc 5:1→EtOAc/MeOH 10:1) and re-crystallization from MeOH thetitle compound as a white solid: TLC (EtOAc) R_(f)=0.50; t_(R)=0.88 min(LC-MS 3); ESI-MS: 484 [M+H]⁺ (LC-MS 3).

Step 24.1:(4S,5R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-aR)-2-((tert-butyldiphenylsilyl)oxy)propyl)-4-methyloxazolidin-2-one

Absolute stereochemistry of the protected 2-hydroxypropyl moiety notdetermined, (R)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor step 22.1 from(4S,5R)-5-((R)-2-((tert-butyldiphenylsilyl)oxy)propyl)-3-(6-chloro-2-morpholinopyrimidin-4-yl)-4-methyloxazolidin-2-oneand intermediate B to afford after purification by flash chromatography(hexane/EtOAc 20:1→EtOAc) the title compound as a light yellow foam: TLC(hexane/EtOAc 1:1) R_(f)=0.45; t_(R)=1.57 min (LC-MS 3); ESI-MS: 722[M+H]⁺ (LC-MS 3).

Step 24.2:(4S,5R)-5-((R)-2-((tert-butyldiphenylsilyl)oxy)propyl)-3-(6-chloro-2-morpholino-pyrimidin-4-yl)-4-methyloxazolidin-2-one

Absolute stereochemistry of the protected 2-hydroxypropyl moiety notdetermined, (R)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor step 21.1 from(4S,5R)-5-((R)-2-((tert-butyldiphenylsilyl)oxy)propyl)-4-methyloxazolidin-2-oneand intermediate A to afford after purification by flash chromatography(hexane/EtOAc 20:1→3:1) the title compound as a colorless oil: TLC(hexane/EtOAc 1:1) R_(f)=0.65; t_(R)=1.67 min (LC-MS 3); ESI-MS: 595,597 [M+H]⁺ (LC-MS 3).

Step 24.3:(4S,5R)-5-((R)-2-((tert-butyldiphenylsilyl)oxy)propyl)-4-methyloxazolidin-2-one

Absolute stereochemistry of the protected 2-hydroxypropyl moiety notdetermined, (R)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor step 23.3 from(4S,5R)-5-((R)-2-((tert-butyldiphenylsilyl)oxy)propyl)-3-(4-methoxybenzyl)-4-methyloxazolidin-2-oneto afford after purification by flash chromatography (hexane/EtOAc20:1→EtOAc) the title compound as a colorless oil: TLC (hexane/EtOAc1:1) R_(f)=0.28; t_(R)=1.38 min (LC-MS 3); ESI-MS: 415 [M+NH₄]⁺ (LC-MS3).

Step 24.4:(4S,5R)-5-((R)-2-((tert-butyldiphenylsilyl)oxy)propyl)-3-(4-methoxybenzyl)-4-methyl-oxazolidin-2-one

Absolute stereochemistry of the protected 2-hydroxypropyl moiety notdetermined, (R)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor step 23.4 from(4S,5R)-5-((R)-2-hydroxypropyl)-3-(4-methoxybenzyl)-4-methyloxazolidin-2-one(contaminated with 15% of the first eluting (2S,4S,5R)-diastereoisomerfrom step 24.5) to afford after purification by flash chromatography(hexane/EtOAc 20:1→EtOAc) the title compound as a colorless oil: TLC(hexane/EtOAc 3:1) R_(f)=0.26; t_(R)=1.55 min (LC-MS 3); ESI-MS: 540[M+Na]⁺ (LC-MS 3).

Step 24.5: (4S,5R)-5-((S)- and(4S,5R)-5-((R)-2-hydroxypropyl)-3-(4-methoxybenzyl)-4-methyl-oxazolidin-2-one

Absolute stereochemistry of 2-hydroxypropyl moiety after reduction ofthe ketone was not determined, the (S)-configuration was arbitrarilyassigned to the first eluting product (4S,5R,5S)-diastereoisomer.

First eluting product is the (4S,5R,5S)-diastereoisomer:

Second eluting product, mixture of (2R,4S,5R)-diastereoisomer(arbitrarily assigned (2R)-configuration for the 2-hydroxypropylmoiety):

contaminated with first eluting (4S,5R,5S)-diastereoisomer:

The title compound was prepared in analogy to the procedure describedfor step 23.5 from a 3:1 mixture of the (4S,5R)- and(4S,5S)-diastereoisomer of3-(4-methoxybenzyl)-4-methyl-5-(2-methylallyl)oxazolidin-2-one to affordafter multiple separations of the 3:3:1:1 mixture of isomers by flashchromatography (RediSep Rf Gold silica gel; hexane/EtOAc 1:1→EtOAc andtoluene/EtOAc 3:1→EtOAc) the individual two minor (2S,4S,5S)- and(2R,4S,5S)-diastereoisomers and the individual two major (2S,4S,5R)- and(2R,4S,5R)-diastereoisomers as colorless oils:

First eluting (2S,4S,5R)-diastereoisomer of the title compound(arbitrarily assigned (2S)-configuration for the 2-hydroxypropylmoiety): TLC (EtOAc) R_(f)=0.39; t_(R)=0.752 min (UPLC 1); t_(R)=0.76min (LC-MS 3); ESI-MS: 280 [M+H]⁺ (LC-MS 3); ¹H NMR (400 MHz, CDCl₃): δ1.12 (d, 3H), 1.28 (d, 3H), 1.5-1.63 (m, 1H), 1.81 (m, 1H), 1.90 (d,1H), 3.68 (m, 1H), 3.82 (s, 3H), 4.03 (d, 1H), 4.14 (m, 1H), 4.60 (ddd,1H), 4.79 (d, 1H), 6.89 (d, 2H), 7.22 (d, 2H).

Second eluting (2R,4S,5R)-diastereoisomer of the title compound(arbitrarily assigned (2R)-configuration for the 2-hydroxypropyl moiety)contaminated with 15% of the first eluting (2S,4S,5R)-diastereoisomer:TLC (EtOAc) R_(f)=0.35; t_(R)=0.742 min and 0.752 min (UPLC 1);t_(R)=0.75 min and 0.76 min (LC-MS 3); ESI-MS: 280 [M+H]⁺ (LC-MS 3); ¹HNMR (400 MHz, CDCl₃): δ 1.12 (d, 3H), 1.28 (d, 3H), 1.50-1.64 (m, 1H),1.81 (m, 0.15H), 1.90 (d, 0.15H). 1.94 (m, 0.85H), 2.21 (d, 0.85H), 3.67(m, 1H), 3.82 (s, 3H), 4.01 (m, 1H), 4.13 (m, 1H), 4.73 (m, 0.85H), 4.79(d, 0.15H), 4.80 (d, 1H), 6.90 (d, 2H), 7.22 (d, 2H).

Step 24.6: (4S,5R)- and (4S,5S)-diastereoisomer of3-(4-methoxybenzyl)-4-methyl-5-(2-methylallyl)oxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 23.6 from a 3:1 mixture of the (2S,3R)- and(2S,3S)-diastereoisomers of benzyl((25)-3-hydroxy-5-methylhex-5-en-2-yl)(4-methoxybenzyl)carbamate toafford after purification by flash chromatography (hexane/EtOAc20:1→1:1) the title compound as a 3:1 mixture of the (4S,5R)- and(4S,5S)-diastereoisomer: t_(R)=1.112 min (UPLC 1); t_(R)=1.05 min (LC-MS3); ESI-MS: 276 [M+H]⁺ (LC-MS 3); ¹H NMR (400 MHz, CDCl₃): δ 1.13 (d,2.2H), 1.20 (d, 0.8H), 1.76 (s, 0.8H), 1.80 (s, 2.2H), 2.23 (ddd,0.25H), 2.27 (dd, 0.75H), 2.39 (dd, 0.25H) 2.46 (dd, 0.75H), 3.28 (m,0.25H), 3.68 (m, 0.75H), 3.83 (s, 3H), 3.99 (d, 0.75H), 4.04 (d, 0.35H),4.14 (m, 0.25H), 4.62 (m, 0.75H), 4.70-4.90 (m, 4H), 6.90 (d, 2H), 7.25(d, 2H).

Step 24.7: (2S,3R)- and (25,35)-diastereomer of Benzyl(3-hydroxy-5-methylhex-5-en-2-yl)(4-methoxybenzyl)carbamate

The title compound was prepared in analogy to the procedure describedfor step 23.7 from (S)-benzyl 4-methoxybenzyl(1-oxopropan-2-yl)carbamateand 3-bromo-2-methylprop-1-ene to afford the title compound as a 3:1mixture of (2S,3R)- and (2S,3S)-diastereomer: t_(R)=1.322 min (majorisomer) and 1.329 min (minor isomer) (UPLC 1); t_(R)=1.23 min (majorisomer) and 1.24 min (minor isomer) (LC-MS 3); ESI-MS: 384 [M+H]⁺ (LC-MS3).

Example 25 Second Eluting Product on the LC MS 3 which is a 9:1 Mixtureof (4S,5R)- and(4S,5S)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-((S)-2-hydroxy-propyl)-4-methyloxazolidin-2-one

Absolute stereochemistry of 2-hydroxypropyl moiety not determined,(S)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor example 23 from a 8:1 mixture of (4S,5R)- and(4S,5S)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-((S)-2-((tert-butyldiphenylsilyl)oxy)propyl)-4-methyloxazolidin-2-oneand TBAF to afford after purification by flash chromatography(hexane/EtOAc 5:1→EtOAc/MeOH 10:1) and re-crystallization from MeOH thetitle compound as a white solid: TLC (EtOAc) R_(f)=0.50; t_(R)=0.87 min(minor (4S,5S)-diastereomer) and 0.89 min (major (4S,5R)-diastereomer)(LC-MS 3); ESI-MS: 484 [M+H]⁺ (LC-MS 3).

Step 25.1: (4S,5R)- and(4S,5S)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-aS)-2-((tert-butyldiphenylsilyl)oxy)propyl)-4-methyloxazolidin-2-one

Absolute stereochemistry of the protected 2-hydroxypropyl moiety was notdetermined, (S)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor step 22.1 from a 7:1 mixture of (4S,5R)- and(4S,5S)-5-((R)-2-((tert-butyldiphenylsilyl)oxy)propyl)-3-(6-chloro-2-morpholinopyrimidin-4-yl)-4-methyloxazolidin-2-oneand intermediate B to afford after purification by flash chromatography(hexane/EtOAc 20:1→EtOAc) the title compound as a 8:1 mixture of(4S,5R)- and (4S,5S)-diastereoisomers: TLC (hexane/EtOAc 1:1)R_(f)=0.45; t_(R)=1.58 min (LC-MS 3); ESI-MS: 722 [M+H]⁺ (LC-MS 3).

Step 25.2: (4S,5R)- and(4S,5S)-54(S)-2-((tert-butyldiphenylsilyl)oxy)propyl)-3-(6-chloro-2-morpholinopyrimidin-4-yl)-4-methyloxazolidin-2-one

Absolute stereochemistry of the protected 2-hydroxypropyl moiety notdetermined, (S)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor step 21.1 from a 7:1 mixture of (4S,5R)- and(4S,5S)-5-((S)-2-((tert-butyldiphenylsilyl)oxy)propyl)-4-methyl-oxazolidin-2-oneand intermediate A to afford after purification by flash chromatography(hexane/EtOAc 20:1→3:1) and crystallization from MeOH/THF the titlecompound as a 7:1 mixture of the (4S,5R)- and (4S,5S)-diastereoisomers:TLC (hexane/EtOAc 1:1) R_(f)=0.65; t_(R)=1.67 min (LC-MS 3); ESI-MS:595, 597 [M+H]⁺ (LC-MS 3).

Step 25.3: (4S,5R)- and(4S,5S)-54(S)-2-((tert-butyldiphenylsilyl)oxy)propyl)-4-methyloxazolidin-2-one

Absolute stereochemistry of the protected 2-hydroxypropyl moiety notdetermined, (S)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor step 23.3 from a 7:1 mixture of (4S,5R)- and(4S,5S)-5-((S)-2-((tert-butyldiphenylsilyl)oxy)propyl)-3-(4-methoxy-benzyl)-4-methyloxazolidin-2-oneto afford after purification by flash chromatography (hexane/EtOAc20:1→2:1) the title compound as a colorless oil as a 10:1 mixture of the(4S,5R)- and (4S,5S)-diastereoisomer: TLC (hexane/EtOAc 1:1) R_(f)=0.30;t_(R)=1.38 min (LC-MS 3); ESI-MS: 415 [M+NH₄]⁺ (LC-MS 3).

Step 25.4: (4S,5R)- and(4S,5S)-54(S)-2-((tert-butyldiphenylsilyl)oxy)propyl)-3-(4-methoxy-benzyl)-4-methyloxazolidin-2-one

Absolute stereochemistry of the protected 2-hydroxypropyl moiety notdetermined, (S)-configuration arbitrary assigned.

The title compound was prepared in analogy to the procedure describedfor step 23.4 from a 7:1 mixture of (4S,5R)- and(4S,5S)-5-((S)-2-hydroxypropyl)-3-(4-methoxybenzyl)-4-methyl-oxazolidin-2-oneprepared in step 24.5 to afford after purification by flashchromatography (hexane/EtOAc 20:1→EtOAc) the title compound as a 7:1mixture of the (4S,5R)- and (4S,5S)-diastereoisomer: TLC (hexane/EtOAc3:1) R_(f)=0.26; t_(R)=1.56 min (LC-MS 3); ESI-MS: 540 [M+Na]⁺ (LC-MS3).

Example 26 (4S,5R)- and(4S,5S)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-hydroxy-2-methylpropyl)-4-methyloxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 22.1 from a 5:1 mixture of (4S,5R)- and(4S,5S)-3-(6-chloro-2-morpholinopyrimidin-4-yl)-5-(2-hydroxy-2-methylpropyl)-4-methyloxazolidin-2-oneand intermediate B to afford after purification by flash chromatography(hexane/EtOAc/MeOH 90:10:1→10:100:10) and re-crystallization from MeOHthe title compound as a white solid and as a 7:1 mixture of the (4S,5R)-and (4S,5S)-diastereoisomer: TLC (EtOAc) R_(f)=0.43; t_(R)=0.93 min(LC-MS 3); ESI-MS: 498 [M+H]⁺ (LC-MS 3).

Step 26.1: (4S,5R)- and(4S,5S)-3-(6-chloro-2-morpholinopyrimidin-4-yl)-5-(2-hydroxy-2-methyl-propyl)-4-methyloxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 21.1 from a 5:1 mixture of (4S,5R)- and(4S,5S)-5-((S)-2-((tert-butyldiphenylsilyl)oxy)propyl)-4-methyloxazolidin-2-oneand intermediate A to afford after purification by flash chromatography(hexane/EtOAc 10:1→EtOAc) the title compound as light yellow foam and asa 5:1 mixture of the (4S,5R)- and (4S,5S)-diastereoisomers: TLC(EtOAc/MeOH 10:1) R_(f)=0.55; t_(R)=1.02 min (LC-MS 3); ESI-MS: 371, 373[M+H]⁺ (LC-MS 3).

Step 26.2: (4S,5R)- and(4S,5S)-5-(2-hydroxy-2-methylpropyl)-4-methyloxazolidin-2-one

The title compound was prepared in analogy to the procedure describedfor step 23.3 from a 5:1 mixture of (4S,5R)- and(4S,5S)-5-(2-hydroxy-2-methylpropyl)-3-(4-methoxybenzyl)-4-methyl-oxazolidin-2-oneto afford after purification by flash chromatography (hexane-EtOAc1:1→EtOAc-MeOH 5:1) the title compound as a colorless oil as a 5:1mixture of the (4S,5R)- and (4S,5S)-diastereoisomer: TLC (EtOAc/MeOH10:1) R_(f)=0.40; t_(R)=0.41 min (LC-MS 3); ESI-MS: 174 [M+H]⁺ (LC-MS3).

Step 26.3: (4S,5R)- and(4S,5S)-5-(2-hydroxy-2-methylpropyl)-3-(4-methoxybenzyl)-4-methyl-oxazolidin-2-one

To a solution of a 3:1 mixture of methyl2-((4S,5R)-3-(4-methoxybenzyl)-4-methyl-2-oxooxazolidin-5-yl)acetate andmethyl2-((4S,5S)-3-(4-methoxybenzyl)-4-methyl-2-oxooxazolidin-5-yl)acetate(2.0 g, 6.82 mmol) in THF (50 mL) was slowly added under argon a 3Mmethylmagnesium chloride solution in THF (6.82 ml, 20.46 mmol) at −78°C. After the addition the reaction mixture was allowed to warm to RT.After addition of 10% aqueous NH4Cl solution the product was extractedwith EtOAc. Combined extracts were washed with H₂O, dried over MgSO₄,filtered and concentrated to afford the title compound afterpurification by flash chromatography (hexane/EtOAc 20:1→EtOAc) as acolorless oil (1.2 g, 59%, 3:1 mixture of (4S,5R)- and(4S,5S)-diastereoisomer): TLC (CH₂Cl₂/MeOH 10:1) R_(f)=0.43; t_(R)=0.80min and 0.81 min (LC-MS 3); ESI-MS: 294 [M+H]⁺ (LC-MS 3); ¹H NMR (400MHz, CDCl₃): δ 1.12 (d, 2.25H), 1.22 (d, 0.75), 1.32 (s, 1.5H), 1.34 (s,4.5H), 1.68 (dd, 1H), 1.86 (dd, 0.25H), 1.93 (dd, 0.75H), 3.23 (m,0.25H), 3.64 (m, 0.75H), 3.83 (s, 3H), 3.99 (d, 0.75H), 4.04 (d, 0.25H),4.26 (d, 0.25H), 4.72 (d, 0.25H), 4.73 (m, 0.75H), 4.80 (d, 0.75H), 6.89(d, 0.5H), 6.90 (d, 1.5H), 7.23 (d, 0.5H), 7.24 (1.5H).

Step 26.4: Methyl24(4S,5R)-3-(4-methoxybenzyl)-4-methyl-2-oxooxazolidin-5-yl)acetate andMethyl24(4S,5S)-3-(4-methoxybenzyl)-4-methyl-2-oxooxazolidin-5-yl)acetate

To a suspension of a 3:1 mixture of the (3R,4S)- and (3S,4S)-methyl3-hydroxy-4-((4methoxybenzyl)amino)pentanoate (4.1 g, 10.74 mmol) inCH₂Cl₂ (80 mL) was added DIEA (8.44 ml, 48.3 mmol) and at 0° C. asolution of (bis(trichloromethyl) carbonate (2.389 g, 8.05 mmol)dissolved in CH₂Cl₂ (10 mL). After stirring for 0.5 h at RT the reactionmixture was added to saturated NaHCO₃ solution and the product wasextracted with CH₂Cl₂. Combined extracts were washed with H₂O, driedover MgSO₄, filtered and concentrated to afford the title compound afterpurification by flash chromatography (hexane/EtOAc 20:1→EtOAc) as alight yellow foam (2.02 g, 63%, 3:1 mixture of the (4S,5R)- and(4S,5S)-diastereoisomers): TLC (toluene/EtOAc 1:1) R_(f)=0.47;t_(R)=0.84 min (LC-MS 3); ESI-MS: 294 [M+H]⁺ (LC-MS 3); ¹H NMR (400 MHz,CDCl₃): δ 1.11 (d, 2.25H), 1.27 (d, 0.75H), 2.57 (dd, 0.25H), 2.67 (dd,0.75H), 2.75 (dd, 0.25H), 2.81 (dd, 0.75H), 3.34 (m, 0.25H), 3.70 (s,0.75H), 3.73 (s, 2.25H), 3.77 (m, 0.75H), 3.83 (s, 3H), 3.99 (d, 0.75H),4.04 (d, 0.25H), 4.44 (m, 0.25H), 4.75 (d, 0.25H), 4.78 (d, 0.75H), 4.88(m, 0.75H), 6.90 (d, 2H), 7.22 (d, 0.5H), 7.23 (d, 1.5H).

Step 26.5: (3R,4S)- and (3S,4S)-Methyl3-hydroxy-4((4-methoxybenzyl)amino)pentanoate

To suspension of (3R,4S)-methyl 4-amino-3-hydroxypentanoatehydrochloride [111061-25-7] (2.17 g, 11.8 mmol) in CH₂Cl₂ (60 mL) andMeOH (60 mL) was added NaOAc (1.357 g, 16.54 mmol) and after 10 minstirring p-anisaldehyde (1.37 mL, 11.2 mmol) and molecular sieve (2 g).The reaction mixture was stirred for 16 h at RT. After the addition of 2mL of AcOH, NaBH₃CN (1.11 g, 17.7 mmol) was added in portions over aperiod of 30 min. After stirring for an additional 30 min at RT, thereaction mixture was filtered, the filtrate acidified with 4N aqueousHCl and evaporated to dryness. The residue was first washed with Et₂O,than suspended in CH₂Cl₂/MeOH 1:1 and the inorganic material wasfiltered off. The filtrate was concentrated to afford the title compoundafter drying at 50° C. for 4 h as a beige solid (2.9 g, 80%, 3:1 mixtureof the (3R,4S)- and (3S,4S)-diastereoisomers): t_(R)=0.46 min((3R,4S)-diastereoisomer) and 0.48 min ((3S,4S)-diastereoisomer) (LC-MS3); ESI-MS: 268 [M+H]+(LC-MS 3).

The ¹H NMR data for the compounds of the above examples is provided inthe following table.

Example 1

¹H NMR (400 MHz, <dmso>) δ ppm 1.41 (d, J = 6.26 Hz, 3 H) 3.67 (dd, J =15.25, 4.30 Hz, 8 H) 4.10 (dd, J = 8.60, 3.13 Hz, 1 H) 4.52 (t, J = 8.21Hz, 1 H) 4.73-4.85 (m, 1 H) 7.41 (s, 1 H) 7.60 (br. s., 2 H) 8.56 (s, 1H) Example 2

¹H NMR (400 MHz, <cdcl₃>) δ ppm 1.52 (s, 3H) 1.61 (s, 3H) 3.76 (s, 8H)3.99 (dd, J = 11.52 Hz, J = 4.10 Hz, 1H) 4.13 (dd, J = 11.52 Hz, J =4.49 Hz, 1H), 4.45 (t, J = 4.30 Hz, 1H) 5.47 (s, 2H) 7.64 (s, 1H) 8.58(s, 1H) Example 3 (racemic)

¹H NMR (400 MHz, <dmso>) δ ppm 1.55 (s, 3H) 3.51 (dd, J = 11.34 Hz, J =5.08 Hz, 1H) 3.65 (s, 8H) 4.05 (d, J = 8.60 Hz, 1H) 4.21 (dd, J = 11.34Hz, J = 5.67 Hz, 1H) 4.39 (d, J = 8.60 Hz, 1H) 5.26 (t, J = 5.47 Hz, 1H)7.41 (s, 1H), 7.59 (br s, 2H), 8.54 (s, 1H) Example 3A Singleenantiomer. Absolute stereo- chemistry not determined.

¹H NMR (400 MHz, <dmso>) δ ppm 1.55 (s, 3H) 3.51 (dd, J = 11.1 Hz, J =5.3 Hz, 1H) 3.65 (s, 8H) 4.05 (d, J = 8.2 Hz, 1H) 4.21 (dd, J = 11.1 Hz,J = 5.7 Hz, 1H) 4.39 (d, J = 8.2 Hz, 1H) 5.26 (t, J = 5.7 Hz, 1H) 7.41(s, 1H) 7.59 (s, 2H) 8.54 (s, 1H) Example 3B Single enantiomer. Absolutestereo- chemistry not determined.

¹H NMR (400 MHz, <dmso>) δ ppm 1.55 (s, 3H) 3.51 (dd, J = 11.1 Hz, J =5.3 Hz, 1H) 3.65 (s, 8H) 4.05 (d, J = 8.2 Hz, 1H) 4.21 (dd, J = 11.1 Hz,J = 5.7 Hz, 1H) 4.39 (d, J = 8.2 Hz, 1H) 5.26 (t, J = 5.7 Hz, 1H) 7.41(s, 1H) 7.59 (s, 2H) 8.54 (s, 1H) Example 4

¹H NMR (400 MHz, <dmso>) δ ppm 1.45 (br. s., 3 H) 1.57-1.96 (m, 3 H)2.13 (d, J = 12.12 Hz, 1 H) 2.77 (br. s., 1 H) 3.65 (br. s., 8 H)3.83-3.95 (m, 1 H) 4.12 (td, J = 11.53, 3.13 Hz, 1 H) 7.11 (s, 1 H) 7.61(br. s., 2 H) 8.56 (s, 1 H) Example 5

¹H NMR (400 MHz, <cdcl3>) δ ppm 3.37-3.41 (m, 3 H) 3.69 (dd, J = 9.37,6.25 Hz, 1 H) 3.72-3.83 (m, 9 H) 4.37-4.55 (m, 2 H) 4.83-4.97 (m, 1 H)5.46 (s, 2 H) 7.60 (s, 1 H) 8.58 (s, 1 H) Example 6

¹H NMR (400 MHz, cdcl3) δ ppm 3.37-3.41 (m, 3H), 3.69 (dd, 1 H),3.72-3.83 (m, 10H), 4.37-4.55 (m, 2H), 4.83-4.97 (m, 1H), 5.46 (s, 2H),7.60 (s, 1H), 8.58 (s, 1H). Example 7

¹H NMR (400 MHz, <dmso>) δ ppm 3.57-3.72 (m, 9 H) 3.81-3.92 (m, 1 H)4.36 (dd, J = 8.60, 3.13 Hz, 1 H) 4.47 (t, J = 8.60 Hz, 1 H) 4.75 (s, 1H) 5.11 (t, J = 5.67 Hz, 1 H) 7.46 (s, 1 H) 7.59 (s, 2 H) 8.54 (s, 1 H)Example 8

¹H NMR (400 MHz, <dmso>) δ ppm 1.49 (d, J = 6.26 Hz, 3 H) 3.67 (dd, J =11.73, 5.08 Hz, 1 H) 3.98 (dt, J = 11.63, 4.35 Hz, 1 H) 4.64 (dd, J =7.23, 3.32 Hz, 1 H) 4.85 (m, J = 6.84 Hz, 1 H) 4.95 (t, J = 4.89 Hz, 1H) 7.48 (s, 1 H) 7.54-7.65 (m, 2 H) 8.54 (s, 1 H) Example 9

¹H NMR (400 MHz, <cd3od>) δ ppm 1.86-2.00 (m, 1H) 2.18-2.34 (m, 1H)3.65-3.85 (m, 10H) 4.38-4.46 (m, 1H) 4.49-4.60 (m, 1H) 4.91- 5.02 (m,1H) 7.51 (s, 1H) 8.53 (s, 1H) Example 10

¹H NMR (400 MHz, <dmso>) δ ppm 1.18 (d, J = 6.65 Hz, 3 H) 1.49 (d, J =6.65 Hz, 3 H) 3.13 (td, J = 13.00, 3.71 Hz, 1 H) 3.33-3.44 (m, 1 H)3.51-3.59 (m, 1 H) 3.62-3.73 (m, 2 H) 3.89 (dd, J = 11.34, 3.13 Hz, 1 H)4.00 (dt, J = 11.53, 4.59 Hz, 1 H) 4.20 (d, J = 11.73 Hz, 1 H) 4.52 (d,J = 5.86 Hz, 1 H) 4.62 (dd, J = 7.43, 2.74 Hz, 1 H) 4.86 (m, 1 H) 4.96(t, J = 4.69 Hz, 1 H) 7.47 (s, 1 H) 7.59 (s, 2 H) 8.54 (s, 1 H) Example11 (for comparison)

¹H NMR (400 MHz, <dmso>) δ ppm 3.56-3.74 (m, 8H) 4.08-4.21 (m, 2H)4.36-4.50 (m, 2H) 7.42 (s, 1H) 4.58 (s, 2H) 8.55 (s, 1H) Example 12

¹H NMR (400 MHz, <dmso>) δ ppm 1.43 (d, J = 6.26 Hz, 3 H) 3.66 (dd, J =15.84, 3.71 Hz, 8 H) 4.39 (d, J = 12.12 Hz, 1 H) 4.69 (dd, J = 12.51,3.52 Hz, 1 H) 4.91-5.04 (m, 2 H) 7.43 (s, 1 H) 7.60 (s, 2 H) 8.22 (s, 1H) 8.56 (s, 1 H) Example 13

¹H NMR (400 MHz, <dmso>) δ ppm 1.12-1.29 (m, 3 H) 1.42 (d, J = 6.26 Hz,3 H) 3.15 (td, J = 13.00, 3.71 Hz, 1 H) 3.35-3.47 (m, 1 H) 3.56 (dd, J =11.53, 2.93 Hz, 1 H) 3.70 (d, J = 11.34 Hz, 1 H) 3.90 (dd, J = 11.34,3.13 Hz, 1 H) 4.10 (dd, J = 8.21, 3.13 Hz, 1 H) 4.22 (d, J = 12.12 Hz, 1H) 4.47-4.59 (m, 2 H) 4.73-4.85 (m, 1 H) 7.39 (s, 1 H) 7.59 (br. s., 2H) 8.55 (s, 1 H) Example 14

¹H NMR (400 MHz, <dmso>) δ ppm 3.50-3.57 (m, 1H) 3.58-3.74 (m, 8H) 3.95(dd, J = 10.3 Hz, J = 6.1 Hz, 1H) 4.16 (dd, J = 10.2 Hz, J = 9.4 Hz, 1H)4.72 (td, J = 6.1 Hz (×2), J = 2.7, 1H) 5.2 (t, J = 5.7, 1H) 7.42 (s,1H) 7.58 (s, 2H) 8.55 (s, 1H) Example 15

¹H NMR (400 MHz, <dmso>) δ ppm 1.32-1.59 (m, 3 H) 3.49-3.76 (m, 10 H)4.23-4.36 (m, 1 H) 4.60 (dt, J = 6.44, 3.42 Hz, 1 H) 5.12-5.28 (m, 1 H)7.41 (t, J = 3.12 Hz, 1 H) 7.59 (br. s., 2H) 8.55 (s, 1 H) Example 16

¹H NMR (400 MHz, <dmso>) δ ppm 1.40 (d, J = 6.25 Hz, 3 H) 3.56-3.79 (m,9 H) 4.29 (dd, J = 10.54, 8.20 Hz, 1 H) 4.74-4.89 (m, 1 H) 7.41 (s, 1 H)7.59 (s, 2 H) 8.54 (s, 1 H) Example 17

¹H NMR (400 MHz, <dmso>) δ ppm 1.41 (d, J = 6.3 Hz, 3H) 4.1 (dd, J = 8.6Hz, J = 3.1 Hz, 1H) 4.52 (t, J = 8.2 Hz, 1H) 4.73-4.87 (m, 1H) 7.40 (s,1H) 7.59 (s, 2H) 8.56 (s, 1H) Example 18

¹H NMR (400 MHz, <dmso>) δ ppm 1.49 (d, J = 6.26 Hz, 3 H) 3.58-3.72 (m,9 H) 3.99 (dt, J = 11.83, 4.64 Hz, 1 H) 4.64 (dd, J = 7.43, 2.74 Hz, 1H) 4.85 (m, 1 H) 4.96 (t, J = 4.69 Hz, 1 H) 7.48 (s, 1 H) 7.59 (s, 2 H)8.54 (s, 1 H) Example 19

¹H NMR (400 MHz, <dmso>) δ ppm 1.30 (d, J = 6.26 Hz, 3 H) 3.59-3.77 (m,10 H) 4.64-4.73 (m, 1 H) 4.85 (t, J = 6.84 Hz, 1 H) 5.15 (t, J = 5.47Hz, 1 H) 7.40 (s, 1 H) 7.59 (s, 2 H) 8.55 (s, 1 H) Example 20

¹H NMR (400 MHz, <dmso>) δ ppm 3.51-3.76 (m, 10 H) 3.95 (dd, J = 10.35,6.05 Hz, 1 H) 4.16 (t, J = 9.76 Hz, 1 H) 4.66-4.82 (m, 1 H) 5.20 (t, J =5.66 Hz, 1 H) 7.42 (s, 1 H) 7.59 (s, 2 H) 8.55 (s, 1 H) Example 21

¹H NMR (600 MHz, DMSO-d₆) δ ppm 3.60-3.75 (m, 8H), 4.12 (m, 4H), 5.15(m, 1H), 5.26 (m, 1H), 7.47 (s, 1H), 7.62 (s, 2H), 8.59 (s, 1H). Example22 1:1 mixture of (3aR,6R,6aR)- and (3aS,6S,6aS)- diastereoisomer

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.04 (d, J = 6.3 Hz, 3H), 1.60-1.75 (m,2H), 1.80-1.95 (m, 1H), 2.24 (m, 1H), 3.60-3.80 (m, 8H), 4.18 (m, 1H),4.72 (d, J = 7.7 Hz, 1H), 5.01 (dt, J = 1.8, 7.3 Hz, 1H), 5.26 (d, J =3.3 Hz, 1H), 7.46 (s, 1H), 7.62 (s, 2H), 8.58 (s, 1H). Example 23

¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.30 (d, J = 6.3 Hz, 3H), 1.87 (m, 2H),3.5-3.75 (m, 10H), 4.73 (t, J = 6.8 Hz, 1H), 4.83 (m, 2H), 7.44 (s, 1H),7.62 (s, 2H), 8.58 (s, 1H). Example 24 first eluting diastereoisomer onthe LC MS 3, (R)-configuration arbitrary assigned.

¹H NMR (400 MHz, CDCl₃) δ ppm 1.26 (d, J = 6.2 Hz, 3H), 1.32 (d, J = 6.3Hz, 3H), 1.75 (m, 1H), 1.96 (m, 1H), 3.41 (d, J = 6.5 Hz, 1H), 3.43 (brd, 1H), 3.69 (m, 8H), 4.03 (m, 1H), 4.72 (m, 1H), 4.80 (m, 1H), 5.38 (m,2H), 7.50 (s, 1H), 8.51 (s, 1H). Example 25 second eluting product onthe LC MS 3, (S)-configuration arbitrary assigned.

¹H NMR (400 MHz, CDCl₃) of 9:1 mixture of (4S,5R)- and (4S,5R)-diastereoisomer δ ppm 1.25 (d, J = 6.2 Hz, 3H), 1.32 (d, J = 6.3 Hz,2.7H), 1.48 (d, J = 6.3 Hz, 0.3H), 1.64 (m, 0.9H), 1.77 (m, 0.1H), 1.86(m, 0.9H), 1.93 (m, 0.1H), 3.70 (m, 8H), 4.06 (m, 2H), 4.37 (m, 0.1H),4.46 (m, 0.1H), 4.77 (m, 0.9H), 4.84 (m, 0.9H), 5.38 (m, 2H), 7.51 (s,1H), 8.51 (s, 1H). Example 26

¹H NMR (400 MHz, CDCl₃) of 10:1 mixture of (4S,5R)- and (4S,5R)-diastereoisomer: δ ppm 1.30 (br s, 8.4H), 1.33 (s, 0.3H)*, 1.56 (0.3H)*,1.79 (d, 1H), 1.95 (dd, 1H), 3.70 (br s, 8H), 4.43 (m, 0.2H)*, 4.70-4.90(m, 1.8H), 5.36 (br s, 2H), 7.51 (s, 1H), 8.51 (s, 1H).Further Physical Properties

The crystalline materials obtained from examples 10 and 18, batches A-Ewere further characterised as follows.

Melting Point Determination:

Melting point was determined by Differential Scanning calorimetry (DSC).DSC was measured using a TA Instruments, DSC 2000, Serial No. 100036. Asample of 1-5 mg was weighed into standard aluminium pan (pan+lid, TA900786.901, 900779.901). The instrument was operated using the ThermalAdvantage Q-Series software V.2.6.0.367 and the Thermal Advantagesoftware V4.6.9. Thermal events were characterized using UniversalAnalysis V4.3A Build 4.3.0.6. The samples was measured against an emptypan. The sample was treated according to the protocol below:

Step 1: EQUILIBRATE AT −40° C.

Step 2: HEAT 10° C./min/300° C.

Modulation: No

The obtained graphs are shown in FIGS. 1, 3, 5, 7, 9 and 11.

Powder X-Ray Diffraction (PXRD):

A sample amount of ca 2-5 mg is placed on an objective glass slide andcentered in the X-ray beam on a Bruker D8 GADDS Discover with CuKa anode(Serial No. 002482). Sample-Detector distance was 30 cm. Two frames wererecorded between 5 and 40° 2-theta. Frames were merged using GADDSsoftware 4.1.27. Evaluation was conducted using EVA 10.0.0.

The obtained graphs are shown in FIGS. 2, 4, 6, 8, 10 and 12.

Representative 2-theta [°] peaks are provided in the following tables:

FIG. 2 (PXRD Example 10)

2-theta [°] Intensity 9.9 medium 14.2 medium 20.1 high 23.2 medium 29.7mediumFIG. 4 (PXRD Example 18A)

2-theta [°] Intensity 8.7 medium 10.6 high 18.5 low 25.3 medium 30.3mediumFIG. 6 (PXRD Example 18B)

2-theta [°] Intensity 10.3 medium 15.2 high 16.0 medium 22.7 medium 23.7mediumFIG. 8 (PXRD Example 18C)

2-theta [°] Intensity 10.3 medium 15.2 high 16.0 medium 20.3 medium 29.0lowFIG. 10 (PXRD Example 18D)

2-theta [°] Intensity 10.3 medium 15.2 high 16.0 medium 16.8 low 20.3mediumFIG. 12 (PXRD Example 18E)

2-theta [°] Intensity 10.2 medium 15.1 high 15.9 medium 20.1 medium 29.0mediumBiological Activity

The efficacy of the compounds of the present invention as PI3 kinaseinhibitors can be demonstrated as follows:

Preparation of Compound Dilutions (384-Well)

Test compounds were dissolved in DMSO (10 mM) and transferred into 1.4mL flat bottom or V-shaped Matrix tubes carrying a unique 2D matrix chipby individual Novartis compound hubs. The numbers of these chips weredistinctively linked to Novartis Pharma Numbers. The stock solutionswere stored at −20° C. if not used immediately. For the test procedurethe vials were defrosted and identified by a scanner whereby a workingsheet was generated that guided the subsequent working steps.

Compounds were either manually diluted in DMSO for individualexperiments (96 wells enabling 10 cpds at 8 (single points)concentrations) as described in or prepared as described below if testedfor profiling in 384-wells. This format enabled the assay of maximally40 individual test compounds at 8 concentrations (single points)including 4 reference compounds. The dilution protocol included theproduction of “pre-dilution plates”, “master plates” and “assay plates”.

Pre-Dilution Plates:

96 polypropylene well plates were used as pre-dilution plates. A totalof 4 pre-dilution plates were prepared including 10 test compounds eachon the plate positions A1-A10, one standard compound at A11 and one DMSOcontrol at A12. The pattern of the dilution steps is summarized inTable 1. Programs have been written to run these pipetting steps on theHamiltonSTAR robots.

TABLE 1 Dilution pattern for pre-dilution plates Vol Vol Conc. (μL) VolConc Dil. Final concentration Row (μL) (μM) DMSO (μL) (μM) ratio (μM) A30 10′000 + 135 → 165 1′820 1:5.5 10 B 50  1′820 + 116 → 166 546 1:3.333 C 50 546 + 100 → 150 182 1:3 1 D 50 182 + 116 → 166 54.6 1:3.33 0.3 E50 54.6 + 100 → 150 18.2 1:3 0.1 F 50 18.2 + 116 → 166 5.46 1:3.33 0.03G 50 5.46 + 100 → 150 1.82 1:3 0.01 H 50 1.82 + 116 → 166 0.546 1:3.330.003 DMSO was saturated with H₂O to a concentration of 10%. Vol:Volume, Conc: Concentration, Dil. ratio: Dilution ratio, Fin. c: Finalconcentration.Master Plates:

100 μL of individual compound dilutions including standard compound andcontrols of the 4 “pre-dilution plates” were transferred into a 384“master plate” including the following concentrations 1,820, 564, 182,54.6, 18.2, 5.46, 1.82 and 0.546 μM, respectively in 90% DMSO.

Assay Plates:

Identical “assay plates” were then prepared by pipetting 50 nL each ofcompound dilutions of the “master plates” into 384-well “assay plates”.The compounds were mixed with 4.5 μL of assays components plus 4.5 μLenzyme corresponding to a 1:181 dilution enabling the finalconcentration of 10, 3.0, 1.0, 0.3, 0.1, 0.03, 0.01 and 0.003 μM,respectively. The preparation of the “master plates” was handled by theMatrix PlateMate Plus robot and replication of “assay plates” by theHummingBird robot.

Method to Generate Expression Constructs

Catalytically active human PI3Kα, PI3Kβ, PI3Kδ, and mTOR were cloned,expressed and purified as described (Maira S M, Stauffer F, Brueggen J,Furet P, Schnell C, Fritsch C, Brachmann S, Chène P, de Pover A,Schoemaker K, Fabbro D, Gabriel D, Simonen M, Murphy L, Finan P, SellersW, García-Echeverría C (2008), Mol Cancer Ther. 7:1851-63 and Maira S M,Pecchi S, Brueggen J, Huh K, Schnell C, Fritsch C, Nagel T, Wiesmann M,Brachmann S, Dorsch M, Chène P, Schoemaker K, De Pover A, Menezes D,Fabbro D, Sellers W, García-Echeverría C, Voliva C F (2011), Mol. CancerTher. accepted).

Biochemical Assays for PI3Kalpha, PI3Kbeta

The luminescence-based ATP detection reagent KinaseGlo was obtained fromPromega, (Cat. No. V6714, Lot No. 236161) through Catalys, Wallisellen,Switzerland. (L-alpha-phosphatidylinositol (PI), Liver, Bovine) wereobtained from Avanti Polar Lipid (Cat. No. 840042C, Lot#LPI-274),Phosphatidylinositol-4,5-bisphosphate (PIP(4,5)₂ (Avanti, Cat. No.840046×) or L-α-phosphatidylinositol (PI) was obtained from Avanti PolarLipid (Cat. No. 840042C, Lot#LPI-274). L-α-Phosphatidylserine (PS) wasfrom Avanti Polar Lipid (Cat. No. 840032C), n-OctylGlucoside AvantiPolar Lipid (Cat. No. 10634425001). Luminescence is a well establishedreadout to determine ATP concentrations and can thus be used to followthe activity of many kinases regardless of their substrate. The KinaseGlo Luminescent Kinase Assay (Promega, Madison/Wis., USA) is ahomogeneous HTS method of measuring kinase activity by quantifying theamount of ATP remaining in solution following a kinase reaction.

50 nL of compound dilutions were dispensed onto black 384-well lowvolume Non Binding Styrene (NBS) plates (Costar Cat. No. NBS#3676) asdescribed in section 8.2. L-α-phosphatidylinositol (PI), provided as 10mg/ml solution in methanol, was transferred into a glass tube and driedunder nitrogen beam. It was then resuspended in 3% OctylGlucoside byvortexing and stored at 4° C. 5 μL of a mix of PI/OG with the PI3Ka andPi3Kb subtypes were added. Kinase reactions were started by addition of5 μl of ATP-mix containing in a final volume 10 μL 10 mM TRIS-HCl pH7.5, 3 mM MgCl₂, 50 mM NaCl, 0.05% CHAPS, 1 mM DTT and 1 μM ATP, andoccurred at room temperature. Reactions were stopped with 10 μl ofKinaseGlo and plates were read 10 mins later in a Synergy2 reader usingan integration time of 0.1 seconds per well. 2.5 μM of NVP-BGT226(standard) was added to the assay plates to generate the 100% inhibitionof the kinase reaction, and the 0% inhibition was given by the solventvehicle (90% DMSO in water). NVP-BGT226 was used as a reference compoundand included in all assay plates in the form of 16 dilution points induplicate.

IC₅₀ values of the percentage inhibition of each compound at 8concentrations (usually 10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and 0.003μM) n=2 were derived by fitting a sigmoidal dose-response curve to aplot of assay readout over inhibitor concentration as described. Allfits were performed with the program XLfit4 (ID Business Solutions,Guildford, UK).

Biochemical Assays for PI3Kdelta, PI3Kgamma

The TR-FRET Adapta™ Universal Kinase Assay Kit was purchased fromInvitrogen Corporation (Carlsbad/CA, USA) (Cat. No. PV5099). The kitcontains the following reagents: Adapta Eu-anti-ADP Antibody (Europiumlabeled anti-ADP antibody in HEPES buffered saline, Cat. No. PV5097),Alexa Fluor® 647-labeled ADP tracer (Alexa Fluor® 647-labeled ADP tracerin HEPES buffered saline, Cat. No. PV5098), proprietary TR-FRET dilutionbuffer pH 7.5 (Cat. No. PV3574).

PIK3CD substrate Phosphatidylinositol was obtained from Invitrogen(vesicules consisting of 2 mM PI in 50 mM HEPES pH7.5; Cat. No. PV5371).PIK3CG substrate Phosphatidylinositol-4,5-bisphosphate (PIP(4,5)₂ wasobtained from Invitrogen (PIP2:PS large unilamellar vesicules consistingof 1 mM PIP2: 19 mM PS in 50 mM HEPES pH7.5, 3 mM MgCl2, 1 mM EGTA; Cat.No. PV5100).

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is atechnology based on energy transfer between two adjacent dyes, from anexcited electron in one dye (the donor) to an electron of an adjacentdye (the acceptor) through resonance, then released as a photon. Thisenergy transfer is detected by an increase in the fluorescence emissionof the acceptor, and a decrease in the fluorescence emission of thedonor. TR-FRET assays for protein kinases use a long-lifetime lanthanideTerbium or Europium chelates as the donor species which overcomeinterference from compound autofluorescence or light scatter fromprecipitated compounds, by introducing a delay after excitation by aflashlamp excitation source. Results are often expressed as a ratio ofthe intensities of the acceptor and donor fluorophores. The ratiometricnature of such a value corrects for differences in assay volumes betweenwells, as well as corrects for quenching effects due to coloredcompounds. The Adapta™ assay can be divided into two phases: a kinasereaction phase and an ADP detection phase. In the kinase reaction phase,all kinase reaction components are added to the well and the reaction isallowed to incubate for a set period of time specific for each kinase.After the reaction, a detection solution of Eu-labeled anti-ADPantibody, Alexa Fluor® 647-labeled ADP tracer, and EDTA (to stop thekinase reaction) are added to the assay well. ADP formed by the kinasereaction will displace the Alexa Fluor® 647-labeled ADP tracer from theantibody, resulting in a decrease in TR-FRET signal. In the presence ofan inhibitor, the amount of ADP formed by the kinase reaction isreduced, and the resulting intact antibody-tracer interaction maintainsa high TR-FRET signal. In the Adapta™ assay, the donor(Europium-anti-ADP antibody) is excited at 340 nm and will transfer itsenergy to the acceptor (Alexa Fluor® 647-labeled ADP tracer). Theemission from the Alexa Fluor® 647 can be monitored with a filtercentered at 665 nm because it is located between the emission peaks ofthe donor, which is measured at 615/620 nm. 50 nL of compound dilutionswere dispensed onto white 384-well small volume polystyrene plate asdescribed in section 2.2. Then 5 μL of PI3Kg and PI3Kd and lipidsubstrate (PI or PIP2:PS) followed by 5 μL of ATP (final assay volume 10μL) are incubated at RT. The standard reaction buffer for the Adapta™TR-FRET assay contained 10 mM Tris-HCl pH 7.5, 3 mM MgCl₂, 50 mM NaCl, 1mM DTT, 0.05% CHAPS. Reactions were stopped with 5 μL of a mixture ofEDTA containing the Eu-labeled anti-ADP antibody and the Alexa Fluor®647-labeled ADP tracer in TR-FRET dilution buffer (proprietary to IVG).Plates are read 15 to 60 mins later in a Synergy2 reader using anintegration time of 0.4 seconds and a delay of 0.05 seconds. Control forthe 100% inhibition of the kinase reaction was performed by replacingthe PI3K by the standard reaction buffer. The control for the 0%inhibition was given by the solvent vehicle of the compounds (90% DMSOin H₂O). The standard compound NVP-BGT226 was used as a referencecompound and included in all assay plates in the form of 16 dilutionpoints in duplicate.

Data are analyzed using Excel fit software or Graphpad Prism. EC₅₀values were derived by fitting a sigmoidal dose-response curve to a plotof assay readout over inhibitor concentration. All fits were performedwith the program XLfit4 (ID Business Solutions, Guildford, UK).Determination of EC₅₀ values of the percentage inhibition of eachcompound at 8 concentrations (usually 10, 3.0, 1.0, 0.3, 0.1, 0.030,0.010 and 0.003 μM) n=2 were derived by fitting a sigmoidaldose-response curve to a plot of assay readout over inhibitorconcentration. All fits were performed with the program XLfit4 (IDBusiness Solutions, Guildford, UK).

Biochemical Assay for mTOR

TR-FRET assays for protein kinases uses a long-lifetime lanthanideTerbium or Europium chelates as the donor species which overcomeinterference by compound autofluorescence or light scatter fromprecipitated compounds, by introducing a delay after excitation by aflashlamp excitation source. Results are often expressed as a ratio ofthe intensities of the acceptor and donor fluorophores. The ratiometricnature of such a value corrects for differences in assay volumes betweenwells, as well as corrects for quenching effects due to coloredcompounds.

Binding Assays are based on the binding and displacement of an AlexaFluor® 647-labeled, ATP-competitive kinase inhibitors to the kinase ofinterest. Invitrogen's “Kinase Tracers” have been developed to address awide range of kinase targets and are based on ATP-competitive kinaseinhibitors, making them suitable for detection of any compounds thatbind to the ATP site or to an allosteric site altering the conformationof the ATP site. Inhibitors that bind the ATP site include both Type Ikinase inhibitors, which bind solely to the ATP site, and Type IIinhibitors (e.g., Gleevec®/Imatinib, Sorafenib, BIRB-796), which bind toboth the ATP site and a hydrophobic site exposed in the DFG-out(non-active) conformation. Type III inhibitors are compounds that do notcompete with ATP are loosely referred to as allosteric inhibitors. Astudy of 15 diverse Type III inhibitors demonstrated that all but onecompound was detected in the binding assay with equivalent potency toactivity assays. The sole exception was a substrate-competitivecompound, and thus not a true allosteric inhibitor.

In contrast to most fluorescence-based kinase activity assays,LanthaScreen® Eu³⁺ Kinase Binding Assays can be read continuously, whichfacilitates evaluation of compounds with slow binding kinetics. Also,unlike most activity assays, binding assays can be performed usingeither active or non-activated kinase preparations, which enablescharacterization of compounds that bind preferentially to non-activatedkinases, such as Gleevec®/Imatinib and some allosteric inhibitors.

In the Lanthascreen™ kinase binding assay, the donor (Eu³⁺-anti-GSTantibody) is excited at 340 nm and will transfer its energy to theacceptor (Alexa Fluor® 647-labeled ATP-competitive kinaseinhibitor=Tracer-314). The emission from the Tracer-314 (Alexa Fluor®647 inhibitor) can be monitored with a filter centered at 665 nm becauseit is located between the emission peaks of the donor, which is measuredat 615/620 nm. The binding of both, the Tracer-314 and Eu³⁺-anti-GSTantibody, to the kinase results in a high degree of FRET from theEu³⁺-donor fluorophore to the Alexa-Fluor® 647-acceptor fluorophore onthe Tracer-314. Binding of an inhibitor to the kinase competes forbinding with the tracer, resulting in a loss of FRET.

50 nL of compound dilutions were dispensed onto white 384-well smallvolume polystyrene plate as described in section 2.2. Then 5 μL ofGST-mTOR and Europium-anti-GST antibody followed by 5 μL of tracer-314(final assay volume 10 μL) are incubated at RT. The standard reactionbuffer for the Lanthascreen™ kinase binding assay contained 50 mM HEPESpH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.01% Pluronic F-127. Plates are read 60mins later in a Synergy2 reader using an integration time of 0.2microseconds and a delay of 0.1 microseconds.

To calculate the emission ratio, the signal emitted at 665 nm from theacceptor (Alexa Fluor® 647-labeled Tracer-314) is divided by the signalemitted at 620 nm from the donor (Eu³⁺ anti-GST antibody)

Control for the 0% inhibition was given by the solvent vehicle of thecompounds (90% DMSO in H₂O). Control for the relative 100% inhibitionwas performed by adding 10 μM in the mix containing GST-mTOR andEuropium anti-GST antibody. An additional control for the absolute 0%inhibition is given by Eu³⁺ anti-GST antibody without GST-mTOR.

Cellular Assays for PI3Kalpha, Beta and Delta

AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay, ALPHA,Perkin Elmer) is a non-radioactive bead-based proximity assay technologyto study biomolecular interactions in a homogenous microtiter plateformat. The brand name SureFire denotes AlphaScreen assays that areadapted to quantify the phosphorylation of endogenous cellular proteinsin cell lysates, by using matched antibody pairs, which consist of ananti-phospho-kinase and an anti-kinase antibody. The assay allowscharacterization of kinase signaling in cells as well as measurement ofkinase inhibitor effects. The AlphaScreen technology provides severaladvantages over standard assay techniques such as ELISA, as it avoidstime-consuming washing procedures and reduces plate handling.Furthermore, it is miniaturizable at least to a 384-well format andprovides sensitivity down to the femtomolar range, dependent on theaffinity of the antibodies included in the individual AlphaScreenSureFire assay kit. High sensitivity is reached by an intrinsicamplification mechanism, which involves production of singlet oxygenmolecules. SureFire assay kits are commercially available for specifictargets and include pairs of validated antibodies (PerkinElmer). Thisreport describes common procedures applied for AlphaScreen SureFireassays and respective semi-automated steps for routine kinase inhibitorprofiling in cell-based assays.

The Rat-1 cell lines stably overexpressing activated PI3K class Iisoforms Rat-1 pBABEpuro Myr-HA-hp110 delta (Rat-1_PI3Kdelta) and Rat-1pBABEpuro Myr-HA-hp110alpha (Rat-1_PI3Kalpha) and Rat-1 pBABEpuroMyr-HA-hp110 beta (Rat-1_PI3beta) were prepared as described (Maira S M,Stauffer F, Brueggen J, Furet P, Schnell C, Fritsch C, Brachmann S,Chène P, de Pover A, Schoemaker K, Fabbro D, Gabriel D, Simonen M,Murphy L, Finan P, Sellers W, García-Echeverría C (2008), Mol CancerTher. 7:1851-63 and Maira S M, Pecchi S, Brueggen J, Huh K, Schnell C,Fritsch C, Nagel T, Wiesmann M, Brachmann S, Dorsch M, Chène P,Schoemaker K, De Pover A, Menezes D, Fabbro D, Sellers W,García-Echeverría C, Voliva C F (2011), Mol. Cancer Ther., accepted).All cell lines were cultivated in complete growth medium (DMEM highglucose, 10% (v/v) fetal bovine serum, 1% (v/v) MEM NEAA, 10 mM HEPES, 2mM L-glutamine, puromycin (10 μg/mL for Rat-1_PI3Kdelta andRat-1_PI3Kalpha, 4 ug/mL for Rat-1_PI3beta), 1% (v/v) Pen/Strep) to 90%confluency at 37° C./5% CO₂/90% humidity in a humidified CO₂ incubatorand were split twice a week.

The following materials were used for p-AKT(S473) detection in Rat-1cell lysates: Dulbecco's modified Eagle's medium (DMEM) high glucose(Gibco Invitrogen, Basel, Switzerland, Cat. No. 41965), Heat InactivatedFetal Bovine Serum, Qualified (HI FBS; Gibco Invitrogen, Basel,Switzerland, Lot. No. 16140), MEM non essential amino acids (NEAA; GibcoInvitrogen, Basel, Switzerland, Cat. No. 11140), HEPES (GibcoInvitrogen, Basel, Switzerland, Cat. No. 15630), Penicillin/Streptomycin(Pen/Strep, 100×; Gibco Invitrogen, Basel, Switzerland, Cat. No.15140-122), L-Glutamine (Gibco Invitrogen, Basel, Switzerland, Cat. No.25030), Puromycin (Sigma Aldrich, Buchs, Switzerland, Cat. No. P9620),DMSO (MERCK, Dietikon, Switzerland, Cat. No. 8.02912.2500), H₂O,MilliQ-H₂O unless otherwise stated (MILLIPORE QGARDOOR1, Millipore, Zug,Switzerland), Bovine serum albumine (BSA; Sigma Aldrich, Buchs,Switzerland Cat. No. A8412), SureFire p-Akt 1/2 (Ser473) Assay Kit(PerkinElmer, Schwerzenbach, Switzerland, Cat. No. TGRAS50K).

The p-Akt(S473) SureFire assay measures the phosphorylation ofendogenous cellular Akt 1/2 at Ser473 in cell lysates. Using Rat-1 cellsstably expressing myr-HA-tagged versions of the human PI3Kdelta,PI3Kalpha, or PI3Kbeta p110 catalytic subunit isoforms, the assay wasdeveloped as a two-plate protocol in a 384-well format.

For compound testing, the cells were seeded at a density of 4000(Rat-1_PI3Kdelta), 7500 (Rat-1_PI3Kalpha), or 6200 (Rat-1_PI3Kbeta)cells in 20 ul complete growth medium into cell culture treated 384-wellplates and were grown at 37° C./5% CO₂/90% humidity for 24 h. Shortlybefore compound transfer, the complete medium was removed, 30 ul assaybuffer (DMEM high glucose, 1×MEM NEAA, 10 mM HEPES, 2 mM L-glutamine,0.1% (w/v) BSA) was added and 10 ul of the compound predilutions weretransferred to the cells. For testing after February 2010, assay bufferwas substituted for complete growth medium, which revealed similarresults (data not shown). After treatment with compound for 1 h, thecells were lysed by the addition of 20 ul lysis buffer supplemented with0.24% (w/v) BSA. Detection of p-AKT(Ser473) was performed with theSureFire p-Akt 1/2 (Ser473) Assay Kit according to the manufacturer'sinstructions using 5 ul of cell lysate in a total detection volume of 12ul.

IC₅₀ values of the percentage inhibition of each compound at 8concentrations (usually 10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and 0.003μM) n=2 were derived by fitting a sigmoidal dose-response curve to aplot of assay readout over inhibitor concentration as described. Allfits were performed with the program XLfit4 (ID Business Solutions,Guildford, UK).

Cellular Assay for mTOR

A cell based assay (384-well format) was developed for determination ofcompound effects on cellular mTOR kinase activity in MEF (mouse embryofibroblasts) cells derived from mice lacking TSC1 (TuberosclerosisComplex1) a potent suppressor of mTOR kinase activity. Due to lack ofTSC1 the mTOR kinase is constitutively activated resulting in permanentphosphorylation of Thr 389 of S6 kinase 1 (S6K1) which is one of thedownstream targets of mTOR.

Using a SureFire Kit that enables to determine the phosphorylation ofThr389 on the S6K1 an assay was developed, validated and implemented inthe Alpha-Screen format that allows the quantitative determination ofphospho-T389 of S6K1 in cell lysates. Treatment of the MEF TSC1−/− cellswith mTOR specific (or mTOR pathway-) inhibitors dose-dependentlyreduced the levels of phospho-T389 on S6K1 allowing calculation of IC50values. These were in agreement with those values obtained with thebiochemical mTOR ATP-binding assay enabling a quantitative comparison ofpotency of mTOR inhibitors.

TSC1−/− MEFs cells (Kwiatkowski, D. J., Zhang, H., Bandura, J. L.,Heiberger, K. M., Glogauer, M., el-Hashemite, N., and Onda, H. (2002)Hum. Mol. Genet. 11, 525-534) were cultured in DMEM high glucose mediumsupplemented with 10% FBS (Invitrogen), 2 mM Glutamine and 1% (w/v)Penicillin/Streptomycin at 37° C., 5% CO₂.

The SureFire kit for determination of P70S6kinase phosphorylation waspurchased from Perkin Elmer (p70S6K p-T389, #TGR70S50K) and the assaywas performed according to the instructions of the supplier andaccording to the generic method for SureFire assays. Shortly, 5 μL celllysate per well were transferred to 384-well white proxy-plates (forluminescent readout) and mixed with 7 μL A and 5 μL B (final volume: 12μL). After 3 h incubation in the dark at RT luminescence was read withthe Envision Reader (Perkin Elmer). Untreated cells were used as control(high control) and cells treated with 3 μM BEZ235 were used as lowcontrol. The assay window between the signals obtained for the high andthe low controls were defined as 100% and compound effects wereexpressed as percent inhibition. IC50 values were calculated from thedose response curves by graphical extrapolation.

The results obtained using the above-described assays are provided inthe following tables, where SEM is the standard error of the mean and nthe number of data measurements.

Biochemical PI3Kalpha PI3Ka/ IC50 Example no. [umol I-1] SEM n 18 0.0090.001 6 15 0.026 0.012 3  1 0.008 0.001 4  4 0.014 — 1 10 0.008 0.002 319 0.006 — 1 13 0.203 — 1 20 0.024 0.003 6  6 0.013 0.006 2  8 0.013 — 1 5 0.011 — 1 16 0.030 0.010 2  7 0.021 — 1 12 0.030 — 1  2 0.023 — 1  30.021 — 1  9 0.384 — 1 11 (comparator) 0.038 0.013 3  3A 0.030 — 1 170.017 — 1 14 0.057 0.020 2  3B 0.031 — 1 21 0.019 — 1 22 0.006 0.001 222A 0.007 0.001 2 22B 0.026 — 1 23 0.006 0.001 3 24 0.005 0.001 2 250.005 —  1* 26 0.005 —  1* WO2007/084786 0.044 0.004 23  Example 10WO2007/084786 0.592 0.076 3 Example 331 WO2007/084786 0.125 — 1 Example17 WO2007/084786 0.359 0.074 3 Example 324 WO2007/084786 0.107 — 1Example 18 WO2007/084786 0.134 0.028 3 Example 344 WO2007/084786 0.0280.012 2 Example 85 *a different, separate measurement gave a value of<0.003 uM.

Biochemical PI3Kbeta PI3Kb/ IC50 Example no. [umol I-1] SEM n 18 0.0040.001 6 15 0.021 0.009 3  1 0.006 0.001 4  4 0.007 — 1 10 0.038 0.025 319 0.005 — 1 13 0.146 — 1 20 0.011 0.002 6  6 0.007 0.003 2  8 0.004 — 1 5 0.011 — 1 16 0.020 0.009 2  7 0.006 — 1 12 0.049 — 1  2 0.020 — 1  30.018 — 1  9 0.041 — 1 11 (comparator) 0.012 0.002 3  3A 0.012 — 1 170.007 — 1 14 0.018 0.003 2  3B 0.032 — 1 21 0.035 — 1 22 0.048 0.038 222A 0.086 0.038 2 22B 0.058 — 1 23 0.011 0.007 2 24 0.016 — 1 25 0.0110.003 2 26 0.124 0.067 2 WO2007/084786 0.165 0.018 22 Example 10WO2007/084786 2.747 0.200 3 Example 331 WO2007/084786 0.214 — 1 Example17 WO2007/084786 1.249 0.468 3 Example 324 WO2007/084786 0.192 — 1Example 18 WO2007/084786 0.875 0.321 3 Example 344 WO2007/084786 0.0370.023 2 Example 85

Biochemical PI3Kdelta PIK3d/ IC50 Example no. [umol I-1] SEM n 18 0.0080.001 5 15 0.015 0.003 3  1 0.007 0.002 4  4 0.004 — 1 10 0.012 0.003 319 0.009 — 1 13 0.021 — 1 20 0.023 0.007 6  6 0.011 0.001 2  8 0.021 — 1 5 0.004 — 1 16 0.021 0.001 2  7 0.017 — 1 12 0.059 — 1  2 0.075 — 1  30.012 — 1  9 0.032 — 1 11 (comparator) 0.029 0.016 3  3A 0.041 — 1 170.008 — 1 14 0.017 0.002 2  3B 0.063 — 1 21 0.017 — 1 22 0.006 0.0025 222A 0.004 0.0020 2 22B 0.005 — 1 23 0.009 0.0003 3 24 0.009 0.0022 2 250.007 0.0025 2 26 0.012 0 2 WO2007/084786 0.236 0.057 11 Example 10WO2007/084786 2.316 0.246 3 Example 331 WO2007/084786 0.296 — 1 Example17 WO2007/084786 0.692 0.037 3 Example 324 WO2007/084786 0.153 — 1Example 18 WO2007/084786 1.039 0.585 2 Example 344 WO2007/084786 0.0800.039 2 Example 85

Biochemical PI3Kgamma PIK3g/ IC50 Example no. [umol I-1] SEM n 18 0.2530.070 6 15 0.338 0.043 3  1 0.276 0.068 4  4 0.238 — 1 10 0.158 0.011 319 0.184 — 1 13 0.386 — 1 20 0.601 0.216 6  6 0.258 0.027 2  8 0.237 — 1 5 0.329 — 1 16 0.770 0.319 2  7 0.529 — 1 12 1.661 — 1  2 0.348 — 1  30.516 — 1  9 1.130 — 1 11 (comparator) 0.998 0.350 3  3A 2.197 — 1 170.328 — 1 14 1.232 0.679 2  3B 1.794 — 1 21 0.370 — 1 22 0.145 0.035 222A 0.110 0.035 2 22B 0.410 — 1 23 0.207 0.087 3 24 0.540 0.340 2 250.205 0.095 2 26 0.290 0.050 2 WO2007/084786 1.898 0.675 11 Example 10WO2007/084786 4.626 * 3 Example 331 WO2007/084786 5.270 — 1 Example 17WO2007/084786 4.322 0.023 3 Example 324 WO2007/084786 8.789 — 1 Example18 WO2007/084786 >9.1 ** 2 Example 344 WO2007/084786 0.464 0.231 2Example 85 * 2 of the 3 values >9.1. No SEM calculation possible. **Both values >9.1. No SEM calculation possible.

Cellular assay PI3Kalpha Rat1-PI3Ka/ IC50 Example no. [umol I-1] SEM n18 0.038 0.011 7 15 0.079 0.026 4  1 0.048 0.006 5  4 0.031 — 1 10 0.0610.013 3 19 0.050 0.007 2 13 0.058 — 1 20 0.158 0.033 5  6 0.094 0.015 3 8 0.081 — 1  5 0.093 — 1 16 0.108 — 1  7 0.120 0.007 2 12 0.113 — 1  20.134 — 1  3 0.147 — 1  9 0.147 — 1 11 (comparator) 0.256 0.076 3  3A0.176 — 1 17 0.178 — 1 14 0.277 0.075 2  3B 0.215 — 1 21 0.085 — 1 220.095 0.009 2 22A 0.086 0.009 2 22B 0.148 — 1 23 0.020 0.007 4 24 0.0450 2 25 0.045 0.013 2 26 0.058 0.031 2 WO2007/084786 0.117 0.012 20Example 10 WO2007/084786 1.770 0.080 2 Example 331 WO2007/084786 0.460 —1 Example 17 WO2007/084786 1.730 0.420 2 Example 324 WO2007/084786 0.669— 1 Example 18 WO2007/084786 1.247 0.035 3 Example 344 WO2007/0847860.126 0.017 4 Example 85

Cellular assay PI3Kbeta Rat1-PI3Kb/ IC50 Example no. [umol I-1] SEM n 180.088 0.035 6 15 0.114 0.010 3  1  0.065* 0.029 3  4 0.183 — 1 10 0.1730.060 4 19 0.071 — 1 13 0.240 — 1 20 0.124 0.032 5  6 0.147 0.128 2  80.108 — 1  5 0.314 — 1 16 0.160 — 1  7 0.132 — 1 12 0.123 — 1  2 0.278 —1  3 0.193 — 1  9 0.226 — 1 11 (comparator) 0.184 0.031 3  3A 0.093 — 117 0.171 — 1 14 0.157 0.026 2  3B 0.331 — 1 21 0.046 — 1 22 0.077 0.0172 22A 0.060 0.017 2 22B 0.125 — 1 23 0.027 0.013 4 24 0.034 0.013 2 250.043 0.009 2 26 0.075 0.037 2 WO2007/084786 0.523 0.047 18  Example 10WO2007/084786 8.220 ** 2 Example 331 WO2007/084786 1.540 — 1 Example 17WO2007/084786 5.310 ** 2 Example 324 WO2007/084786 1.580 — 1 Example 18WO2007/084786 4.035 0.540 4 Example 344 WO2007/084786 0.229 0.049 4Example 85 *fourth measurement 3.9 uM, outlier ** The second valuewas >10. Therefore no SEM calculation was possible.

Cellular assay PI3Kdelta Rat1-PI3Kd/ IC50 Example no. [umol I-1] SEM n18 0.028 0.006 7 15 0.053 0.020 4  1 0.027 0.005 4  4 0.013 — 1 10 0.0520.011 3 19 0.034 0.010 2 13 0.042 — 1 20 0.111 0.017 5  6 0.037 0.003 3 8 0.054 — 1  5 0.023 — 1 16 0.087 — 1  7 0.070 0.015 3 12 0.092 — 1  20.081 — 1  3 0.073 — 1  9 0.078 — 1 11 (comparator) 0.138 0.050 3  3A0.059 — 1 17 0.155 — 1 14 0.101 0.008 2  3B 0.172 — 1 21 0.061 — 1 220.027 — 1 22A 0.027 — 1 22B 0.005 — 1 23 0.016 0.002 4 24 0.007 0 4 250.013 0.002 5 26 0.011 0.004 4 WO2007/084786 0.548 0.034 20 Example 10WO2007/084786 5.220 * 2 Example 331 WO2007/084786 1.030 — 1 Example 17WO2007/084786 6.540 0.040 2 Example 324 WO2007/084786 1.290 — 1 Example18 WO2007/084786 2.768 0.184 4 Example 344 WO2007/084786 0.238 0.065 4Example 85 * The second value was >10. Therefore no SEM calculation waspossible.

Cellular assay mTOR mTOR S6K(T389)_TSC1ko/ Example no. IC50 [umol I-1]SEM n 18 0.794 0.070 7 15 0.727 * 2  1 0.964 0.228 3  4 0.647 0.013 2 102.020 ** 4 19 0.423 0.050 2 13 1.950 — 1 20 >2.27 8  6 0.574 0.100 4  80.994 — 1  5 1.41 0.05  2 16 0.942 4  7 1.23 0.09  2 12 1.100 — 1  30.759 0.048 2  9 1.260 * 2 11 (comparator) 1.410 *** 5  3A 1.570 0.170 217 0.867 — 1 14 >2.27 **** 3  3B 1.111 0.230 2 21 0.942 0.025 2 22 1.3860.734 2 22A 2.120 0.730 2 22B 2.3 — 1 23 1.111 0.320 2 24 1.830 — 1 251.830 — 1 26 0.857 — 1 WO2007/084786 0.602 0.067 10 Example 10WO2007/084786 >2.27 **** 4 Example 331 WO2007/084786 2.120 * 2 Example17 WO2007/084786 >2.27 **** 4 Example 324 WO2007/084786 >2.27 **** 2Example 18 WO2007/084786 >2.27 **** 3 Example 344 WO2007/084786 1.0010.055 3 Example 85 * the second value was >2.27. Therefore no SEMcalculation was possible. ** 3 of the 4 values were >2.27. Therefore noSEM calculation was possible. *** 3 of the 5 values were >2.27.Therefore no SEM calculation was possible. **** All values were >2.27.Therefore no SEM calculation was possible.

The off-target effect (evidence of tubulin binding) was measured asfollows.

Cytospin Assay Description:

Cell cycle G2/M arrest Cytospin assay to detect tubulin binding(off-target) binding activities of MAPP derivatives: 5×10⁵ cells A2058cells were plated in 6-well cluster, with 2 mL of DMEM (high Glucosecontaining 1% sodium pyruvate, 1% glutamin and 10% FCS). 18 hours later,the test items were added at a concentration of 5 μM (spiking 1 μL of a10 mM solution of the test item). 24 hours later, the cells weretrypsinized and transferred into a 15 mL conical tube. Cells are thenpelleted by centrifugation, and resuspended with PBS/0 (containing 10%FCS). Cells are counted with a CASY counter, and each samplesequilibrated to 1×10⁶ cells/mL. 200 μL (2×10⁵ cells) were thentransferred to 1.5 mL Cytospin tubes (Heraeus Sepatec, Ref 1152), andcentrifuged for 5 min at 50×g at 4° C., with a Cytospin system,containing a Sepatech system (Heraeus, Ref #3425), adjusted on top of amicroscope slide (Thermo-Scientific, Ref:#PH040820. Cells were thenfixed for 15 min at room temperature, and stained with the Diff Quick®assay (Medion Diagnostics, Ref:#130832), following the recommendationsof the manufacturer. Condensed DNA reflecting the G2/M arrest isrevealed by punctuated staining in the cells, when examining the slidesunder the microscope. The staining was visually assessed for thepresence of condensed DNA and given a score where 0=no condensed DNAobserved (indicating no off target activity), 1=(indicating weak offtarget activity), 2=(indicating medium off target activity), 3=largeamount of condensed DNA observed (indicating strong off-targetactivity).

The data obtained using this method is shown in the following table:

Example no. Score 18 0 15 0  1 0  4 0 10 0 19 0 13 0 20 1-2  6 0  8 0  50 16 0  7 n.d. 12 0  2 n.d.  3 0  9 n.d. 11 (comparator) 3  3A 0 17 0 140  3B 0 21 0 22 0 22A n.d. 22B n.d. 23 0 24 0 25 0 26 0 WO2007/084786 3Example 10 WO2007/084786 0 Example 331 WO2007/084786 0 Example 17WO2007/084786 0 Example 324 WO2007/084786 0 Example 18 WO2007/084786 0Example 344 WO2007/084786 3 Example 85 n.d. = not done

What is claimed is:
 1. A compound of Formula (I)

wherein, R¹ is

wherein R^(1a) is H or —CH₃ or R¹ is

wherein D is deuterium; R² is H; R³ is H; R⁴ is H, and R⁵ is —CH₃ or—CH₂OH; or R⁴ is —CH₂OH, and R⁵ is H; or R² is —CH₃, —CH₂OH, —CH₂OCH₃,—CH₂CH₂OH or —CH₂OC(O)H; R³ is H; R⁴ is —CH₃, —CH₂OH, —CH₂CH₂OH,—CH₂CH(OH)CH₃ or —CH₂C(OH)(CH₃)₂ and R⁵ is H, or R⁴ is H, and R⁵ is—CH₃, —CH₂OH, —CH₂CH(OH)CH₃ or —CH₂C(OH)(CH₃)₂, or R⁴ is H or —CH₃ andR⁵ is H or —CH₃; or R³ is H; R⁴ is H; R² and R⁵ are joined and form—(CH₂)₄—; or R⁴ is H; R⁵ is H; and R² is —CH₂OH, and R³ is —CH₃; or R²is H or —CH₃, and R³ is —CH₂OH; or R² is H; R⁴ is H; and R³ and R⁵ arejoined and form the group

or the group

or R³ is H; R⁵ is H; and R² and R⁴ are joined and form the group

or a pharmaceutically acceptable salt thereof.
 2. The compound accordingto claim 1, wherein, R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or—CH₂OC(O)H; R³ is H; R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, or R⁴is H, and R⁵ is —CH₃ or —CH₂OH, or R⁴ is H or —CH₃ and R⁵ is H or —CH₃;or R³ is H; R⁴ is H; R² and R⁵ is —(CH₂)₄—; or R⁴ is H; R⁵ is H; and R²is —CH₂OH, and R³ is —CH₃; or R² is H or —CH₃, and R³ is —CH₂OH, or apharmaceutically acceptable salt thereof.
 3. The compound according toclaim 1, wherein, R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H;R³ is H; R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, or R⁴ is H, andR⁵ is —CH₃ or —CH₂OH, or R⁴ is H or —CH₃ and R⁵ is H or —CH₃; or R⁴ isH; R⁵ is H; and R² is —CH₂OH, and R³ is —CH₃; or R² is H or —CH₃, and R³is —CH₂OH, or a pharmaceutically acceptable salt thereof.
 4. Thecompound according to claim 1, wherein, R² is —CH₃, —CH₂OH, —CH₂OCH₃,—CH₂CH₂OH or —CH₂OC(O)H; R³ is H; R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, andR⁵ is H, or R⁴ is H, and R⁵ is —CH₃ or —CH₂OH, or R⁴ is H or —CH₃ and R⁵is H or —CH₃, or a pharmaceutically acceptable salt thereof.
 5. Thecompound according to claim 1, of formula (IA′)

wherein R^(1a) is H or —CH₃ or a pharmaceutically acceptable saltthereof.
 6. The compound according to claim 1, of formula (IA):

wherein, R² is —CH₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH or —CH₂OC(O)H; R³ is H;R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵ is H, or R⁴ is H, and R⁵ is —CH₃or —CH₂OH, or R⁴ is H or —CH₃ and R⁵ is H or —CH₃, or a pharmaceuticallyacceptable salt thereof.
 7. The compound according to claim 6, whereinR² is —CH₃ or —CH₂OH; R³ is H; R⁴ is —CH₃, —CH₂OH or —CH₂CH₂OH, and R⁵is H, or R⁴ is H, and R⁵ is —CH₃ or —CH₂OH, or R⁴ is H or —CH₃ and R⁵ isH or —CH₃, or a pharmaceutically acceptable salt thereof.
 8. Thecompound according to claim 7, wherein R² is —CH₃ or —CH₂OH; R³ is H; R⁴is —CH₃, —CH₂OH or —CH₂CH₂OH and R⁵ is H or R⁴ is H and R⁵ is CH₃ or—CH₂OH, or a pharmaceutically acceptable salt thereof.
 9. The compound,or a pharmaceutically acceptable salt thereof, according to claim 1which is selected from(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-methyl-oxazolidin-2-one,(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5,5-dimethyl-oxazolidin-2-one,racemic3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-4,5′-bipyrimidin-6-yl)-4-(hydroxymethyl)-4-methyloxazolidin-2-one,(S)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-4,5′-bipyrimidin-6-yl)-4-(hydroxymethyl)-4-methyloxazolidin-2-one(absolute stereochemistry not determined),(R)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-4,5′-bipyrimidin-6-yl)-4-(hydroxymethyl)-4-methyloxazolidin-2-one(absolute stereochemistry not determined),(3aS,7aS)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-hexahydro-benzooxazol-2-one,(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-methoxymethyl-oxazolidin-2-one,(4S,5S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one,(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-oxazolidin-2-one,(4S,5R)-3-(2′-Amino-2-(D8-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one,(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-(2-hydroxy-ethyl)-oxazolidin-2-one,(4S,5R)-3-[2′-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-hydroxymethyl-5-methyl-oxazolidin-2-one,Formic acid(4S,5R)-3-(2′-amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-methyl-2-oxo-oxazolidin-4-ylmethylester,(S)-3-[2′-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-methyl-oxazolidin-2-one,(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-oxazolidin-2-one,(4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-4-methyl-oxazolidin-2-one,(S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-methyl-oxazolidin-2-one,(S)-3-(2′-amino-2-D8-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-4-methyloxazolidin-2-one,(4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one,(4S,5S)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-4-methyl-oxazolidin-2-one,(R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-5-hydroxymethyl-oxazolidin-2-one,(3aR,6aR)-3-(2′-amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-tetrahydrofuro[3,4-d]oxazol-2(3H)-one,racemic(3aR*,6R*,6aR*)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-hydroxyhexahydro-2H-cyclopenta[d]oxazol-2-one,(3aR,6R,6aR)-(Z-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-hydroxyhexahydro-2H-cyclopenta[d]oxazol-2-one,(3aS,6S,6aS)-(Z-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-6-hydroxyhexa-hydro-2H-cyclopenta[d]oxazol-2-one,or(4S,5R)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-hydroxyethyl)-4-methyloxazolidin-2-one.10. A compound, or a pharmaceutically acceptable salt thereof, selectedfrom(4S,5R)-3-[2′-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl]-4-hydroxymethyl-5-methyl-oxazolidin-2-one,(4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one,or(4S,5R)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-hydroxyethyl)-4-methyloxazolidin-2-one.11. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound according to claim 1, or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptablecarriers.
 12. A combination comprising a therapeutically effectiveamount of a compound according to claim 1, or a pharmaceuticallyacceptable salt thereof, and one or more additional therapeuticallyactive agents.
 13. A method of treating in a patent with cancer selectedfrom a group consisting of HER2 positive breast cancer, solid tumors andnon-small cell lung cancer comprising administering to a subject atherapeutically effective amount of a compound, or a pharmaceuticallyacceptable salt thereof, according to claim
 1. 14. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundaccording to claim 9, or a pharmaceutically acceptable salt thereof, andone or more pharmaceutically acceptable carriers.
 15. A combinationcomprising a therapeutically effective amount of a compound according toclaim 9, or a pharmaceutically acceptable salt thereof, and one or moreadditional therapeutically active agents.
 16. A method of treating in apatient with cancer selected from the group consisting of HER2 positivebreast cancer, solid tumors and non-small cell lung cancer comprisingadministering to a subject a therapeutically effective amount of acompound, or a pharmaceutically acceptable salt thereof, according toclaim
 9. 17. A pharmaceutical composition comprising a therapeuticallyeffective amount of a compound according to claim 10, or apharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable carriers.
 18. A combination comprising atherapeutically effective amount of a compound according to claim 10, ora pharmaceutically acceptable salt thereof, and one or more additionaltherapeutically active agents.
 19. A method of treating in a patientwith cancer selected from the group consisting of HER2 positive breastcancer, solid tumors and non-small cell lung cancer comprisingadministering to a subject a therapeutically effective amount of acompound, or a pharmaceutically acceptable salt thereof, according toclaim
 10. 20. A compound(4S,5R)-3-[2′-Amino-2-((S)-3-methyl-morpholin-4-yl)-4′-trifluoromethyl-[4,5′]bipyrimidinyl]-6-yl]-4-hydroxymethyl-5-methyl-oxazolidin-2-one.21. A compound(4S,5R)-3-(2′-Amino-2-morpholin-4-yl-4′-trifluoromethyl-[4,5′]bipyrimidinyl-6-yl)-4-hydroxymethyl-5-methyl-oxazolidin-2-one.22. A compound(4S,5R)-3-(2′-Amino-2-morpholino-4′-(trifluoromethyl)-[4,5′-bipyrimidin]-6-yl)-5-(2-hydroxyethyl)-4-methyloxazolidin-2-one.